Engineered human extracellular dnase enzymes for drug candidate selection

ABSTRACT

The present disclosure provides a library of engineered DNASE proteins (including DNASE1, DNASE1-LIKE 1, DNASE1-LIKE 2, DNASE1-LIKE 3, DNASE2A, DNASE2B) that allows to select drug candidates for developing therapeutics for treating conditions characterized by neutrophil extracellular trap (NET) accumulation and/or release. In accordance with the invention, the selected DNase variant has improved properties, including properties amenable to clinical development, including manufacturing, toxicology, pharmacokinetic, and/or use in therapy.

PRIORITY

This application claims the benefit of, and priority to, U.S.Application No. 62/800,790, filed Feb. 4, 2019, which is herebyincorporated by reference in its entirety.

BACKGROUND

Inflammation is an essential host response to control invading microbesand heal damaged tissues. Uncontrolled and persistent inflammationcauses tissue injury in a plethora of inflammatory disorders.Neutrophils are the predominant leukocytes in acute inflammation. Duringinfections neutrophils generate neutrophil extracellular traps (NETs),lattices of DNA-filaments decorated with toxic histones and enzymes thatimmobilize and neutralize bacteria. However, excessive NET formation mayharm host cells due to their cytotoxic, proinflammatory, andprothrombotic activity.

DNASE1 (D1) forms along with DNASE1-LIKE 1 (D1L1), DNASE1-LIKE 2 (D1L2)and DNASE1-LIKE 3 (D1L3), the DNASE1-protein family, a group ofhomologous secreted DNase enzymes. DNASE2A and DNASE2B form anadditional group of homologous extracellular DNase enzymes. DNASE1- andDNASE2-protein family members are evolutionary conserved and expressedin various species, including humans. Recombinant human DNASE1- andDNASE2-protein family members provide drug candidates for NET-associateddiseases, but the physical, enzymatic, toxicological, andpharmacokinetic properties of these enzymes are not ideal for clinicalapplications. Thus, there is a need for engineered DNASE enzymes for usein therapy that have improved properties, including properties amenableto clinical development, including manufacturing, toxicology,pharmacokinetic, and/or use in therapy.

SUMMARY

The present invention provides candidates of engineered humanextracellular DNASE proteins (e.g., variants of DNASE1 (D1), DNASE1-LIKE1 (D1L1), DNASE1-LIKE 2 (D1L2), DNASE1-LIKE 3 Isoform 1 (D1L3),DNASE1-LIKE 3 Isoform 2 (D1L3-2), DNASE2A (D2A), and DNASE2B (D2B)) thatare useful for treating conditions characterized by extracellular DNA,extracellular chromatin, and/or neutrophil extracellular trap (NET)accumulation and/or release. In accordance with aspects of theinvention, DNASE variants described herein are more amenable to clinicaldevelopment, including manufacturing, toxicology, pharmacokinetic,and/or use in therapy.

In some aspects, the invention provides a method for making a DNASEtherapeutic composition for treating an extracellular chromatin orNET-associated disorder. The method comprises evaluating a plurality ofextracellular DNASE variants for one or more characteristics, includingenzymatic activity, nucleic acid substrate preference, potential forrecombinant expression in prokaryotic or eukaryotic host cells,immunogenic potential in humans and animals, and pharmacodynamics inanimal models. An extracellular DNASE variant is selected with thedesired enzymatic, physical, and pharmacodynamics profile, and isformulated for administration to a patient, e.g., for either systemic orlocal administration.

In various embodiments, the DNASE variant evaluated and selected for usein therapy in accordance with embodiments of the invention comprise anamino acid sequence that is at least 80% identical to the enzyme definedby any one of SEQ ID NOS: 1 to 7, with one or more building blocksubstitutions or C-terminal modifications as described herein. In someembodiments, the DNASE variant comprises an N-terminal or C-terminalfusion to a half-life extending moiety, such as albumin, transferrin, anFc, or elastin-like protein.

In various embodiments, a selected DNASE variant is formulated with apharmaceutically acceptable carrier for systemic, local, or topicaladministration.

In other aspects, the invention provides a method for treating a subjectin need of extracellular DNA degradation, extracellular chromatindegradation, extracellular trap (ET) degradation and/or neutrophilextracellular trap (NET) degradation. The method comprises administeringa therapeutically effective amount of the extracellular DNASE variant inaccordance with the disclosure.

Other aspects and embodiments of the disclosure will be apparent fromthe following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the approach for engineering DNASE variants fortherapeutic applications using Building Block Protein Engineering.

FIG. 2 shows an alignment of DNASE1-LIKE 3 Isoform 1 proteins fromdifferent species. Amino acids that are non-conserved in human DNASE1are highlighted. Such non-conserved amino acids can be transferred tohuman DNASE1-LIKE 3 Isoform 1 for developing a variant for therapy. TheDNASE1-LIKE 3 Isoform 1 proteins used for this analysis were HumanDNASE1-LIKE 3 Isoform 1, UniProtKB: Q13609; NCBI Reference Sequence: NP004935.1 (SEQ ID NO: 4); Pan troglodytes (Chimpanzee) DNase1L3UniProtKB: A0A213RHL6 (and H2QMU7) (SEQ ID NO: 33); Papio anubis (Olivebaboon) DNASE1L3, UniProtKB: A0A213NFJ3 (SEQ ID NO: 34); Mouse Dnase113,UniProtKB: 055070 (SEQ ID NO: 31); Rat DNase1L3, UniProtKB: 089107 (SEQID NO: 32); Oryctolagus cuniculus (Rabbit) DNase1L3, UniProtKB: G1SE62(SEQ ID NO: 35); Canis lupus familiaris (Dog) DNase1L3, UniProtKB:F1P9C1 (SEQ ID NO: 36); Sus scrofa (Pig) DNase1L3, UniProtKB: A0A287B132(SEQ ID NO: 37); Cavia porcellus (Guinea pig) DNase1L3, UniProtKB:A0A286XK50 (SEQ ID NO: 38); Bos taurus (Cow) DNase1L3, UniProtKB: F1MGQ1(SEQ ID NO: 39); and Loxodonta africana (African elephant) DNase1L3,UniProtKB: G3SXX1 (SEQ ID NO: 40)

FIG. 3 shows an alignment of two members of the human DNASE1 proteinsfamily, DNASE1-LIKE 1 and DNASE1-LIKE 3 Isoform 1. Amino acids that areconserved among human DNASE1-LIKE 1 (NCBI Reference Sequence: NP006721.1; SEQ ID NO: 2) and DNASE1-LIKE 3 Isoform 1 (NCBI ReferenceSequence: NP 004935.1; SEQ ID NO: 4) are highlighted. The non-conservedamino acids can be transferred from human DNASE1-LIKE 1 to DNASE1-LIKE 3Isoform 1 or vice versa for developing variants for therapy,respectively.

FIG. 4 shows the concept of building block engineering of homologousproteins. The technology transfers single or multiple variable aminoacids, which are flanked by conserved single or multiple variable aminoacids, between a donor and recipient protein.

FIG. 5 shows an amino acid sequence alignment of DNase1 and DNase1L3 ofmouse (SEQ ID NOs: 31 and 32), rat (SEQ ID NOs: 33 and 34), chimpanzee(SEQ ID NOs: 35 and 36), and human (SEQ ID NOs: 1 and 4). The N-terminalsignal peptide, corresponding to N-terminal 22 amino acids of DNase1 isshown in light grey and conserved amino acids are highlighted in adarker shade of grey. Variable amino acids are not highlighted and serveas Building Blocks that can be transferred from DNase1 to DNase1L3 andvice versa. Abbreviations: AA, amino acid.

FIGS. 6A-FIG. 6B show lists of Building Blocks in human DNase1 (D1) andhuman DNase1L3 (D1L3). FIG. 6A shows amino acids that are conserved inD1 and D1L3, which serve as N- and C-anchors, respectively. Buildingblocks are variable amino acids in D1 and D1L3. Mutations that transferBuilding Blocks from D1L3 to D1 are shown. FIG. 6B shows N- andC-anchors in D1L3. Mutations that transfer Building Blocks from D1 toD1L3 are listed. AA: amino acid.

FIG. 7 shows an application of the building block engineering ofhomologous proteins. The application uses as an initial screening step,the transfer of clusters of building blocks between a homologous donorand recipient protein. Additional optional steps are the transfer ofindividual building blocks, followed by the transfer of individual aminoacids. In a final step (not shown), multiple amino acids, buildingblocks, and building block clusters may be combined to degenerate achimeric enzyme.

FIG. 8 shows characterization of DNase1 variants (D1^(V)) featuringbuilding blocks from DNase1L3 (D1L3). Zymography showed dsDNA degradingactivity as dark circles. The dsDNA degrading activity correlates withthe diameter. Samples without activity show the loading well as smallblack spot (e.g. Ctrl). Agarose gel electrophoresis (AGE) of DNAisolated from digested chromatin shows a shift from high-molecularweight DNA to lower or low-molecular weight DNA that correlates withchromatin degrading activity. Building block substitutions that cause anincrease in chromatin degrading activity are highlighted in dark shade.Samples without such effect are shown in light shade. A DNase1 variantfeaturing the combination of building blocks 11, 12-14, 26, 41-48, and49 shows similar chromatin degrading activity than wild-type DNase1L3.

FIG. 9 illustrates that the mutation Q282 S305delinsK in D1L3 Isoform 1increases the activity to degrade high-molecular weight chromatin ofDNASE1L3.

DETAILED DESCRIPTION

As used herein, the term “neutrophil extracellular trap” or “NET” refersto any extracellular trap (“ET”) comprising extracellular DNA formed bycells such as, but not limited to, neutrophils, monocytes, macrophages,basophils, eosinophils, mast cells, cancer cells, injured cells (e.g.,injured endothelial cells), and the like. Unless the context indicatesotherwise, the terms NET and ET are used interchangeably herein.

The similarity of nucleotide and amino acid sequences, i.e. thepercentage of sequence identity, can be determined via sequencealignments as known in the art. Such alignments can be carried out withseveral art-known algorithms, such as with the mathematical algorithm ofKarlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA90: 5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/)or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson,T. J. (1994) Nucleic Acids Res. 22, 4673-80). Exemplary algorithms areincorporated into the BLASTN and BLASTP programs of Altschul et al(1990) J. Mol. Biol. 215: 403-410. When utilizing BLAST programs, thedefault parameters of the respective programs are used.

In various aspects, the invention provides a protein engineeringtechnology that is based on a transfer of a single amino acid ormultiple-adjacent amino acids, termed “building block”, between twomembers of a protein family, such as DNase1 or DNase 2 protein familymembers to generate enzymatically active variants. A “building block” isdefined by amino acids that are variable between two or more members ofthe DNase protein family. These variable amino acids are flanked byamino acids that are conserved between two or more members of theDNase-protein family (“anchors”). The variable single amino acid ormultiple contiguous amino acids (“building blocks”) are exchangedbetween members of the DNase-protein family by implanting them betweenconserved single amino acid or multiple contiguous amino acids(“anchors”).

This approach is referred to herein as “building-block proteinengineering.” Where three or more amino acids are transferred in abuilding block, up to ⅓ of the amino acids transferred may be furthersubstituted. For example, where three to six amino acids are transferredas a building block, one or up to two resides may be furthersubstituted. In some embodiments, four or more amino acids aretransferred as a building block substitution, and up to 25% of thetransferred amino acids are further substituted, e.g., with conservativeor non-conservative amino acid modifications. For example, where four,eight, or twelve amino acids are transferred, one, two, or three aminoacids (respectively) may be further substituted in the building blocksubstitution.

The present invention provides candidates of engineered humanextracellular DNASE proteins (e.g., variants of DNASE1 (D1), DNASE1-LIKE1 (D1L1), DNASE1-LIKE 2 (D1L2), DNASE1-LIKE 3 Isoform 1 (D1L3),DNASE1-LIKE 3 Isoform 2 (D1L3-2), DNASE2A (D2A), and DNASE2B (D2B)) thatare useful for treating conditions characterized by extracellular DNA,extracellular chromatin, and/or neutrophil extracellular trap (NET)accumulation and/or release. In accordance with aspects of theinvention, DNASE variants described herein are more amenable to clinicaldevelopment, including manufacturing, toxicology, pharmacokinetic,and/or use in therapy.

In some aspects, the invention provides a method for making a DNASEtherapeutic composition for treating a NET-associated disorder ordisorder characterized by pathological accumulation of extracellularchromatin. The method comprises evaluating a plurality of extracellularDNASE variants for one or more characteristics, including enzymaticactivity, nucleic acid substrate preference, suitability for recombinantexpression in prokaryotic or eukaryotic host cells, immunogenicpotential in humans and animals, and pharmacodynamics in animal models.An extracellular DNASE variant is selected with the desired enzymatic,physical, and pharmacodynamics profile, and is formulated foradministration to a patient, e.g., for either systemic or localadministration.

In various embodiments, at least 5 or at least 10, or at least 20, or atleast 50 extracellular DNASE variants are evaluated, with the variantsselected from one or more of D1 variants, D1L1 variants, D1L2 variants,D1L3 isoform 1 variant, D1L3 isoform 2 variants, D2A variants, and D2Bvariants as described herein. As described herein, one or more (or all)variants may comprise at least one building block substitution,half-life extension moiety, and/or other mutation or variation describedherein. In some embodiments, the method evaluates one or more D1L1variants described herein. In some embodiments, the method evaluates oneor more D1L2 variants described herein. In some embodiments, the methodevaluates one or more D1L3 variants described herein. In someembodiments, the method evaluates one or more D1L3-2 variants describedherein. In some embodiments, the method evaluates one or more D2Avariants described herein. In some embodiments, the method evaluates oneor more D2B variants described herein. In some embodiments, the methodevaluates one or more D1 variants described herein.

In various embodiments, the invention provides a recombinant variant ofhuman extracellular DNASE enzymes comprising one or more amino acidalterations resulting in an altered pH and temperature optimum,requirement for divalent cations for enzymatic activity, mechanisms ofenzymatic inhibition (e.g. salt, divalent cations, actin, heparin,proteases), substrate affinity and specificity (e.g. single-strandedDNA, double-stranded DNA, chromatin, NETs, plasmid DNA, mitochondrialDNA), localization upon secretion (e.g. membrane-bound, extracellularmatrix), localization signals (e.g. nuclear localization signal,membrane anchor), glycosylation sites, disulfide-bonds and unpairedcysteines, compatibility with GMP-compliant in vitro expression systems(e.g. bacteria. yeast, mammalian cells), compatibility with carriers(e.g. PEGylation, Fc fragment, albumin), compatibility withGMP-compliant purification methods (e.g. anion exchange resins, cationexchange resins), toxicological profile, tissue penetration,pharmacokinetics and pharmacodynamics. In accordance with thisdisclosure, candidate DNASE variants can be selected with desiredproperties for therapy.

In various embodiments, DNASE variants will comprise at least onebuilding block substitution, using a Building Block Protein Engineeringtechnology. The Building Block Engineering approach is described inPCT/US2018/047084, which is hereby incorporated by reference in itsentirety. This approach involves providing a protein-protein alignmentof donor and recipient DNASE enzymes, and identifying variable aminoacid sequences for transfer (“building block”). The variable aminoacid(s) are flanked by one or more conserved amino acids in the donorand recipient DNASE enzymes (upstream and downstream of the buildingblock). These building blocks can be swapped between recipient and donorproteins, to produce a chimeric enzyme. The donor and recipient DNASEenzymes can be selected from members of the DNASE1- or DNASE2-proteinfamily. Accordingly, for example, human DNASE1 and human DNASE1L1 can beselected as donor and recipient DNASE proteins, respectively.Alternatively, donor and recipient DNASE can be selected from a DNASEproteins that are expressed in different species. Accordingly, forexample, bovine and human DNASE1 can be selected as donor and recipientDNASE proteins, respectively.

As used herein, when referring to sequence identity with wild-typeextracellular DNASE enzymes, and unless stated otherwise, sequencesrefer to mature enzymes lacking the signal peptide. Further, unlessstated otherwise, amino acid positions are numbered with respect to thefull translated extracellular DNASE sequence, including signal peptide,for clarity. Accordingly, for example, reference to sequence identity tothe enzyme of SEQ ID NO: 1 (human D1) refers to a percent identity withthe mature enzyme having L23 at the N-terminus, reference to sequenceidentity to the enzyme of SEQ ID NO: 2 (human D1L1) refers to a percentidentity with the mature enzyme having F19 at the N-terminus, referenceto sequence identity to the enzyme of SEQ ID NO: 3 (human D1L2) refersto a percent identity with the mature enzyme having L22 at theN-terminus, reference to sequence identity to the enzyme of SEQ ID NO: 4(human D1L3) refers to a percent identity with the mature enzyme havingM21 at the N-terminus, reference to sequence identity to the enzyme ofSEQ ID NO: 5 (human D1L3-2) refers to a percent identity with the matureenzyme having M21 at the N-terminus, reference to sequence identity tothe enzyme of SEQ ID NO: 6 (human D2A) refers to a percent identity withthe mature enzyme having C19 at the N-terminus, and reference tosequence identity to the enzyme of SEQ ID NO: 7 (human D2B) refers to apercent identity with the mature enzyme having A28 at the N-terminus.

The term “delins” refers to a deletion between two indicated aminoacids, with an insertion of an amino acid or sequence of amino acids atthe site of the deletion. For example, the notation E91_P92delinsSRmeans that the amino acids from E91 to P92 are deleted and the aminoacids SR are inserted at the site of the deletion (e.g., the resultingamino acid sequence will have S91 and R92).

The term “ins” refers to an insertion of amino acids between twoindicated amino acids. For example, the notation E91_P92insSR means thatthe amino acids SR are inserted between E91 and P92, resulting in thesequence E91, S92, R93, and P94.

The term “del” refers to a deletion of one amino acid or two and moreamino acids between two indicated amino acids. For example, the notationE91del means that the amino acid E91 is deleted, whereas the notionE91_P93del means that the three amino acids from E91 and P93 aredeleted.

The engineered variants of human extracellular DNASE enzymes maycomprise one or more additional amino acid substitutions, additions(insertions), deletions, or truncations in the amino acid sequence ofthe human enzyme (SEQ ID NO: 1 to 7). Amino acid substitutions mayinclude conservative and/or non-conservative substitutions. For example,“conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, size, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the amino acid residuesinvolved. The 20 naturally occurring amino acids can be grouped into thefollowing six standard amino acid groups: (1) hydrophobic: Met, Ala,Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3)acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influencechain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.“Conservative substitutions” are defined as exchanges of an amino acidby another amino acid listed within the same group of the six standardamino acid groups shown above. For example, the exchange of Asp by Gluretains one negative charge in the so modified polypeptide. In addition,glycine and proline may be substituted for one another based on theirability to disrupt α-helices. As used herein, “non-conservativesubstitutions” are defined as exchanges of an amino acid by anotheramino acid listed in a different group of the six standard amino acidgroups (1) to (6) shown above.

In various embodiments, the DNASE1 (D1) variant evaluated and selectedfor use in therapy in accordance with embodiments of the inventioncomprises an amino acid sequence that is at least 80% identical to theenzyme defined by SEQ ID NO: 1, with one or more building blocksubstitutions.

In some aspects, the building block substitutions are selected fromnon-human D1 proteins and result in variants of human D1, which featureone or more of the following mutations: K24R, I25M, Q31R, T32S, E35D,V44S, V44T, V44A, S45V, S45K, S45N, S45H, Q49K, Q49R, S52Q, S52R, R53L,I56V, A57V, L58V, V59I, S60T, T68V, D75N, N76E, N76K, N76T, N76E, N76Y,N76S, Q79R, Q79E, D80K, D80H, A81K, A81I, A81D, P82A, P82T, D83N, D83G,T84N, T84A, Y85F, H86R, Y87F, Y87H, V88I, V89I, V89A, N96K, N96R, N96S,S97T, R101Q, V105L, Y106F, D109S, Q110R, Q110K, A113V, A113I, S114L,S116T, Y108Q, Y108H, Y108L, P125S, N128T, T130S, N132S, N132A, A136S,I137V, R139K, F141S, F141H, S142C, R143P, R143H, F144Y, F144S, F144L,V147K, V147Q, R148Q, R148S, E149K, I152V, P154A, A157S, G160E, G160T,G160L, G160S, D161E, V163A, S164S, D167N, A168S, D175N, Q177W, Q177R,E178Q, E178K, E178H, G181D, G181H, E183Q, E183N, V185I, M186V, L187F,G194D, C195Y, R199T, R199A, R199S, P200S, P200A, P200T, P200L, Q202H,S204A, W209R, T210M, T210E, P212S, T213A, T213I, T213P, Q215K, Q215R,P219L, S221T, S221N, A226V, A226S, T227S, T227K, P228S, H230N, A232P,M241T, M241A, M241P, M241S, R244Q, G245D, G245A, G245H, G245R, G245S,D250N, D250S, D250E, D250G, L253V, L253A, L253M, N256D, A259V, A260E,Y261F, G262R, S264T, D265N, D265S, D265E, Q266E, L267M, L267T, Q269E,Q269L, M280T, M280A, K282R, K282A, K282T, K282insK, and K282insR.

In some embodiments, the building block substitutions to D1 are selectedfrom human D1L1 and result in variants of human D1 which feature one ormore of the following mutations: M1_G3del, K5_G8delinsHYPT, A12F,L14_Q15delinsAN, V21_K24delinsQAFR, A26C, I30A, T32 T36delinsRLTLA,M38_I47delinsVAREQVMDTL, Q49R, S52A, Y54C, A57 V59_delinsMVL, R63V,H66_T68delinsSGS, V70_K72delinsIPL, D75_N76delinsRE, Q79delinsRF,A81_T84delinsGSGP, H86_V89delinsSTLS, E91_P92delinsPQ, N96_S97delinsST,K99M, R101T, L103_V105delinsVYF, P108_D115delinsSHKTQVLS, Y118V, D120N,G122_N128delinsED, T130V, A136_R139delinsFVAQ,F141_I152delinsSLPSNVLPSLVL, A157_A158delinsTT, G160_A164delinsKAVEK,I166_D167delinsLN, Y173F, D175E, G177_E179delinsQSK, M186L, G194D,S196_R207delinsASLTKKRLDKLE, W209R, S211E, T213G, Q215H, L217V, P219A,S221_A222delinsGE, A226_P228delinsVRAS, A232T, I236V, V238_A239delinsLH,M241_L243delinsERC, G245_S251delinsSLLHT, L253_P254delinsAA, N256D,Q259_G262delinsPTSQG, S264_L267delinsTEEE, Q269_A270delinsLN, M280E, andK282delinsKLSQAHSVQPLSLTVLLLLSLLSPQLCPAA.

In certain embodiments, the building block substitutions to D1 areselected from human D1L2 and result in variants of human D1 whichfeature one or more of the following mutations: R2G, M4_KSdelinsPRA,G8A, L11W, A14E, L16_Q18delinsA, A20_S22delinsTAA, K24R, A26G, T32S,E35_T36delinsDS, M38V, N40_V44delinsDPACG, Y46I, V48_Q49delinsAK,S52_R53delinsAG, I56L, S65_H66delinsPD, T68S, S70_A71delinsGK,L74_L77delinsMEQI, Q79_T84delinsSVSEHE, H86_Y87delinsSF, V89S, E91Q,D95_Q96delinsNS, R101M, P108K, Q110A, A113V, S116T, Y118L, P119D,G122_N128delinsPE, T130V, N132S, A136_I137delinsFV, R139K,F141_F144delinsSAPG, E146_V153delinsGERAPPLPSRRALTPPLPAAAQNLVLI,G160_D161delinsHQ, Q177_E178delinsID, L182_E182delinsTD,V185_M188delinsMLFL, G194D, P200_Q202delinsAQD, S204_S205delinsAA,W209_T210delinsRS, P212_T213delinsEV, Q230K, A226_H230delinsVGNSD,V239_A240delinsAC, M241_L242delinsAR, G245_V248delinsRSLK, D250Q,L253_N256delinsTVHD, A259_Y262delinsEEF, S264_L267delinsDQTQ, Q269L,Y275F, M280T, and K282insFHR.

In some embodiments, the building block substitutions to D1 are selectedfrom human DNASE1-LIKE 3 Isoform 1 (D1L3) and result in variants ofhuman D1, which feature one or more of the following mutations:M1_L6delinsMSRE, G8_A9deinsAP, A12L, A14_G19delinsLLSIHS,V21_K24delinsLAMR, A26_A27delinsCS, I30_T32delinsVRS, S36T,M38_Y46delinsQEDKNAMDV, Q49_S52delinsKVIK, Y54C, A57I, Q60M,V62_R63delinsIK, H66_K72delinsNNRICPI, L74_N76delinsMEK,Q79_D83delinsRNSRRGI, H86N, V89I, E91_P92delinsSR, S97T, R101Q, L103A,V105L, R107_Q110delinsKEKL, A113_D115delinsVKR, Y118H, D120H,G122_N128delinsYQDGDA, T130V, N132S; A136_I137delinsFV, R139W, F141Q,R143_F144delinsPH, E146A, R148_E149delinsKD, A151V, V153I,A157_A158delinsTT, G160_A162delinsETS, A164K, A168E, Y170_D171 delinsVE,L174T, Q177_K179delinsKHR, G181_L182delinsKA, D184_L187delisNFIF,R199_Q202delisPKKA, S204_S205delinsKN, W209R, S211D, T213R, Q215V,P219G, S221_A222delinsQE, A226_P228delinsVKKS, H230N, V238_A239delinsLR,M241_A246delinsQEIVSS, D250K, A252_P254delinsNSV, N256D, A259K, G262K,S264_L267delinsTEEE, Q269_I271delinsLDV, Y275F, V279_M280delinsFK, andK282delinsQSSRAFTNSKKSVTLRKKTKSKRS.

In some embodiments, the building block substitutions to D1 are selectedfrom human DNASE1-LIKE 3 Isoform 2 (D1L3-2) and result in variants ofhuman D1, which feature one or more of the following mutations: M1L6delinsMSRE, G8_A9deinsAP, A12L, A14_G19delinsLLSIHS,V21_K24delinsLAMR, A26_A27delinsCS, I30_T32delinsVRS, T36S,M38_Y46delinsQEDKNAMDV, Q49_S52delinsKVIK, Y52C, A57I, Q60M,V62_R63delinsIK, H66_K72delinsNNRICPI, L74_N76delinsMEK, Q79_Y106del,P108_Q110delinsEKL, A113_D115delinsVKR, Y118H, D120H,G122_N128delinsYQDGDA, T130V, N132S, A136_I137delinsFV, R139W, F141Q,R143 F144delinsPH, E146A, R148_E149delinsKD, A151V, V153I,A157_A158delinsAA, G160_A162delinsETS, A164K, Y170_D171 delinsVE, L174T,Q177_K179delinsKHR, G181_L182delinsKA, D184_L187delisNFIF,R199_Q202delisPKKA, S204_S205delinsKN, W209R, S211D, T231R, Q215V,P219G, S221_S222delinsQE, A226_P228delinsVKKS, H230N, V238_A239delinsLR,M241_A246delinsQEIVSS, D250K, A252_P254delinsNSV, N256D, G262K,S264_L267delinsTEEE, Q269_I271delinsLDV, Y275F, V279_M280delinsFK, andK282delinsQSSRAFTNSKKSVTLRKKTKSKRS.

In various embodiments, the D1 variant evaluated in accordance with thedisclosure comprises the D1 wildtype amino acid sequence or a variantsequence described herein, fused to the C-terminus of a carrier protein,with a linking amino acid sequence. In some embodiments, the carrierprotein is Fc fragment or albumin. In some embodiments, the carrierprotein is human albumin. The linking sequence can be a flexible linkerpredominately composed of Gly, or Gy and Ser. In some embodiments, thelinker is composed of Gly and amino acids having hydrophilic side chains(e.g., Ser, Thr). In some embodiments, the linker is from 5 to 20 aminoacids. An exemplary linker has the structure (GGGGS)₃.

In various embodiments, the peptide linker may be a flexible linker, arigid linker, or in some embodiments a physiologically-cleavable linker(e.g., a protease-cleavable linker). In some embodiments, the linker is5 to 100 amino acids in length, or is 5 to 50 amino acids in length.

Linkers, where present, can be selected from flexible, rigid, andcleavable peptide linkers. Flexible linkers are predominately orentirely composed of small, non-polar or polar residues such as Gly, Serand Thr. An exemplary flexible linker comprises (Gly_(y)Ser)_(n)linkers, where y is from 1 to 10 (e.g., from 1 to 5), and n is from 1 toabout 10, and in some embodiments, is from 3 to about 6. In exemplaryembodiments, y is from 2 to 4, and n is from 3 to 8. Due to theirflexibility, these linkers are unstructured. More rigid linkers includepolyproline or poly Pro-Ala motifs and α-helical linkers. An exemplaryα-helical linker is A(EAAAK)_(n)A, where n is as defined above (e.g.,from 1 to 10, or 2 to 6). Generally, linkers can be predominatelycomposed of amino acids selected from Gly, Ser, Thr, Ala, and Pro.Exemplary linker sequences contain at least 10 amino acids, and may bein the range of 15 to 35 amino acids. Exemplary linker designs areprovided as SEQ ID NOS: 41 to 51.

In some embodiments, the variant comprises a linker, wherein the aminoacid sequence of the linker is predominately glycine and serineresidues, or consists essentially of glycine and serine residues. Insome embodiments, the ratio of Ser and Gly in the linker is,respectively, from about 1:1 to about 1:10, from about 1:2 to about 1:6,or about 1:4. Exemplary linker sequences comprise S(GGS)₄GSS (SEQ ID NO:46), S(GGS)₉GS (SEQ ID NO: 47), (GGS)₉GS (SEQ ID NO: 48). In someembodiments, the linker has at least 10 amino acids, or at least 15amino acids, or at least 20 amino acids, or at least 25 amino acids. Forexample, the linker may have a length of from 15 to 30 amino acids. Invarious embodiments, longer linkers of at least 15 amino acids canprovide improvements in yield upon expression in Pichia pastoris.

In other embodiments, the linker is a physiologically-cleavable linker,such as a protease-cleavable linker. For example, the protease may be acoagulation pathway protease, such as activated Factor XII. In certainembodiments, the linker comprises the amino acid sequence of Factor XI(SEQ ID NO: 49) and/or prekallikrein (SEQ ID NO: 50 or 51) or aphysiologically cleavable fragment thereof. In other embodiments, thelinker includes a peptide sequence that is targeted for cleavage by aneutrophil specific protease, such as neutrophil elastase, cathepsin G,and proteinase 3.

In various embodiments, the human DNASE1-LIKE 1 (D1L1) variant evaluatedand selected for use in therapy in accordance with embodiments of theinvention comprise an amino acid sequence that is at least 80% identicalto the enzyme defined by SEQ ID NO: 2, with one or more building blocksubstitutions.

In some embodiments, the building block substitutions to D1L1 areselected from non-human D1L1 proteins and result in variants of humanD1L1 which feature one or more of the following mutations: A26T, Q27H,A32T, A32S, V34L, A35T, A35I, R36K, Q38S, Q38E, Q38H, Q38Y, Q38P, Q38D,M40K, M40L, T42I, L43F, R45Q, R45K, L47V, M53T, S61A, S62T, G63Q, G63D,G63N, G63S, S64N, S64A, S64K, S64T, A65T, P66L, P66S, L68F, R71Q, R71E,E72K, N74S, R75K, F76Y, D77K, D77Q, D77Y, D77G, G78A, G78S, G78N, G78D,G80R, G80K, P81S, P81F, P81C, S83R, T84F, T84S, L85H, S86N, S86K, P88S,P88D, Q89L, Q89M, S93N, S93G, T94A, M96V, M96K, T98K, V100A, F102I,H106D, K107R, K107E, K107R, T108A, Q109E, V110L, L111R, S112N, S112D,S112E, S113F, V115Q, V115L, V115M, N117D, N117E, N117P, N177S, E119T,E119Q, E119K, V122I, V122L, A124T, A130G, A130C, Q131H, Q131W, S133T,L134F, P135R, N137D, N137K, V138T, V138I, L142V, V143A, A153D, K156P,K156N, K156T, L159K, Y162H, D163E, D163T, E167D, V168A, S169Y, S169A,Q170R, Q170G, H171R, S174N, S174T, K175E, K175Q, S176N, V177M, V177I,A188T, T191A, T191N, D196K, D196N, D196S, D196A, D196G, E199L, E199A,E199V, E203K, E203D, E203Q, P204A, P204V, P204T, H207R, H207S, V209A,I210V, A211P, E214D, E241V, H223N, T225A, V229I, L231V, L231M, E234Q,E234V, R235G, R235T, R235L, C236L, R237Q, S238M, S238K, S238G, L240M,H241K, H241Q, H241S, H241R, T242A, T242S, T242N, T242G, A244T, D247N,T240K, T250R, Q250Q, S251T, S251R, Q252R, Q252G, T255N, T255S, E258Q,E259Q, N261R, N261K, M261T, I262V, E271D, K273S, K273N, K273D, K273A,L274del, S275Q, S275K, S275R, Q276A, A277T, A277V, H278P, H278Q, S279G,S279N, S279R, C279S, I280V, A280V, Q281P, Q281L, L283H, L283P, S284Y,S284C, S284H, S284G, T286A, T286S, T286V, V287T, V287A, F287F, G287V,L288A, L288S, L289S, L289V, L289M, S292L, S292P, S295P, S295T, S295A,P296S, Q297E, L298C, C299D, C299G, C299S, P300L, A301Q, A301V, andA302M.

In some embodiments, the building block substitutions to D1L1 areselected from human D1 and result in variants of human D1L1 whichfeature one or more of the following mutations: MldelinsMRGM,H2_T5delinsKLLG, F9A, A13_N14delinsLQ, Q17_R20delinsVLSK, C22A, A261,R28_A32delinsTFGET, V34_L43delinsMSNATLVSYI, R45Q, A48S, C50Y,M53_L55delinsALV, V59R, S62_S64delisHLT, I66_L68delinsVGK,R71_E72delinsDN, R75_F76delinsQ, G78_P81delinsAPDT, S83_S86delinsHYVV,P88_Q89delinsEP, S93_T94delinsNS, M96K, T98R, V100_F102delinsLFV,S105_S112delinsPDQVSAVD, V115Y, N117D, E119_D120delinsGCEPCGN, V122T,F128_Q131delinsAIVR, S133_L144delinsFSRFTEVREFAI, T149_T150delinsAA,K152_K156delinsGDAVA, L158_N159delinsID, F165Y, E167D,Q173_K175delinsGLE, M188L, D186G, A188_E199delinsSYVRPSQWSSIR, R201W,E203S, G205T, H207Q, V209L, A211P, G213_E214delinsSA,V218_S221delinsATP, T225A, V229I, L23I_H232delinsVA, E234_C236delinsMLL,S238_T242delinsGAVVPDS, A244_A245delinsLP, D247N, P249_Q253delinsQAAYG,T255_E258delinsSDQL, L260_N261delinsQA, E271M, and L274_A302del.

In some embodiments, the building block substitutions to D1L1 areselected from human D1L2 and result in variants of human D1L1 whichfeature one or more of the following mutations: H2_Y3delinsGG, TSR,F9_L12delinsWALE, N14A, A16_Q17delinsTA, F19L, C22G, A261,R28_A32delinsSFGDS, A35_R45delinsSDPACGSIIAK, R49_50CdelinsGY,I52_L55delinsLALV, V59R, S61_G63delinsPDL, I66_L68delinsVSA,L70_L73delinsMEQI, R75_P81delinsSVSEHE, T84_L85delinsFV,P88_Q89delinsQP, S93_T94delinsDQ, M96K, T98M, V100_F102delinsLFV,S105_Q109delinsKDAVS, L111_S113delinsVDT, V115L, N117P,E119_P120delinsPE, A124S, A130_Q131delinsVK, L134A,S136_V138delinsGTGERAPP, S141_L142insRRALTPPPLPAAAQN, V145I,T149_T150delinsAA, K152_K156delinsHQAVA, L158_N159delinsID, F165Y, E167D, S169_H171delinsIDK, V177_L179delinsMLF,A188_E199delinsSYVRAQDWAAIR, T202_G205delinsSSEV, H207K, V209L, A211P,G213_E214delinsSA, R219_H223delinsGNSD, T225A, V229I, L231_H232delinsAC,E234A, C236L, S238_L239delinsRS, H241_T242delinsKPQS,A244_F246delinsTVH, P249_S251delinsQEE, Q253G, T255_E258delinsDQTQ,N261A, Y266F, E271T, and L274_A302delinsFHR.

In some embodiments, the building block substitutions to D1L1 areselected from human DNASE1-LIKE 3 Isoform 1 (D1L3) and result invariants of human D1L1, which feature one or more of the followingmutations: H2 TSdelinsSREL, L7_L8insPLL, F9L, I11_G14delinsLSIHS, Q17L,F19M, A23S, A26_A32delinsVRSFGES, V34_V39delinsQEDKNA, T42_L43delinsVI,R45_A48delinsKVIK, M53_Q56delinsILVM, V58_V59delinsIK,S62_I66delinsNNRIC, L68I, L70_E72delinsMEK, F76_S79delinsNSRR,G80_P81delinsIT, S83_S86delinsNYVI, P88_Q89delinsPQ, S92N, M96K, T98Q,V100_F102delinsAFL, R104_Q109delinsKEKLVS, L111_S112delinsKR, V115H,N117H, E119_D120delinsYQDGDA, A124S, A130_Q131delinsVW,S133_L134delinsQS, S136_L142delinsHTAVKDF, L144_V145delinsII,K152_E155delinsETSV, L158_A160delinsIDE, Y162_D163delinsVE,F165_E167delinsVTD, S169_H171delinsKHR, Q173_V177delinsKAENF, D186G,A188_T191delinsSYVP, R194_E199delinsAWKNIR, E203D, G205R, H207V, V209L,A211G, G213Q, R219_A220delinsKK, H223N, T225A, V229I, H232R,E234_R237delinsQEIV, L239_A245delinsSVVPKSNSV, P249_Q253delinsQKAYK,N261_I262delinsDV, Y266F, V270_E271delinsFK, K273_L274delinsQS, Q276R,H278_L283delinsFTNSKK, L285V, and V287_A302delinsLRKKTKSKRS.

In some embodiments, the building block substitutions to D1L1 areselected from human DNASE1-LIKE 3 Isoform 2 (D1L3-2) and result invariants of human D1L1, which feature one or more of the followingmutations: H2_T5delinsSREL, A7delinsPLL, F9L, I11_G14delinsLSIHS, Q17L,F19M, S23A, A26_A32delinsVRSFGES, V34_V39delinsQEDKNA, T42_L43delinsVI,R45_A48delinsKVIK, M53_Q56delinsILVM, V58_V59delinsIK,S62_I66delinsNNRIC, L68I, L70_E72delinsMEK, F75_Q103del,S105_Q109delinsEKLVS, L111_S112delinsKR, V115H, N117H,E119_D120delinsYQDGDA, A124S, A130_Q131delinsVW, S133_L134delinsQS,S136_L142delinsHTAVKDF, L144_V145delinsII, K152_E155delinsETSV,L158_A160delinsIDE, Y162_D163delinsVE, F165_E167delinsVTD, S169_H171delinsKHR, Q173_V177delinsKAENF, D186G, A188_T191delinsSYVP,R194_E199delinsAWKNIR, E203D, G205R, H207V, V209L, A211G, G213Q,R219_A220delinsKK, H223N, T225A, V229I, H232R, E234_R237delinsQEIV,L239_A245delinsSVVPKSNSV, P249_Q253delinsQKAYK, N261_I262delinsDV,Y266F, V270_E271delinsFK, K273_L274delinsQS, Q276R,H278_L283delinsFTNSKK, L285V, and V287_A302delinsLRKKTKSKRS.

In certain embodiments, the D1L1 protein variant contains one or moreamino acid substitutions, additions, or deletions in the C-terminal taildomain defined by SEQ ID NO: 8. The C-terminal tail domain or a portionthereof may be deleted. In some embodiments, at least 3, 5, 8, 10, 12,15, 18, or 23 amino acids of the C-terminal tail domain are deleted.

In various embodiments, the D1L1 variant evaluated in accordance withthe disclosure comprises the D1L1 wildtype amino acid sequence or avariant sequence described herein, fused to the C-terminus of a carrierprotein, with a linking amino acid sequence. In some embodiments, thecarrier protein is Fc fragment or albumin. In some embodiments, thecarrier protein is human albumin. The linking sequence, which is alsoherein referred to as a peptide linker, or a linker, can be a flexiblelinker predominately composed of Gly, or Gy and Ser. In someembodiments, the linker is composed of Gly and amino acids havinghydrophilic side chains (e.g., Ser, Thr). In some embodiments, thelinker is from 5 to 20 amino acids. An exemplary linker has thestructure (GGGGS)₃.

The peptide linker may be a flexible linker, a rigid linker, or in someembodiments a physiologically-cleavable linker (e.g., aprotease-cleavable linker). In some embodiments, the linker is 5 to 100amino acids in length, or is 5 to 50 amino acids in length.

Linkers, where present, can be selected from flexible, rigid, andcleavable peptide linkers. Flexible linkers are predominately orentirely composed of small, non-polar or polar residues such as Gly, Serand Thr. An exemplary flexible linker comprises (Gly_(y)Ser)_(n)linkers, where y is from 1 to 10 (e.g., from 1 to 5), and n is from 1 toabout 10, and in some embodiments, is from 3 to about 6. In exemplaryembodiments, y is from 2 to 4, and n is from 3 to 8. Due to theirflexibility, these linkers are unstructured. More rigid linkers includepolyproline or poly Pro-Ala motifs and α-helical linkers. An exemplaryα-helical linker is A(EAAAK)_(n)A, where n is as defined above (e.g.,from 1 to 10, or 2 to 6). Generally, linkers can be predominatelycomposed of amino acids selected from Gly, Ser, Thr, Ala, and Pro.Exemplary linker sequences contain at least 10 amino acids, and may bein the range of 15 to 35 amino acids. Exemplary linker designs areprovided as SEQ ID NOS: 41 to 51.

In some embodiments, the variant comprises a linker, wherein the aminoacid sequence of the linker is predominately glycine and serineresidues, or consists essentially of glycine and serine residues. Insome embodiments, the ratio of Ser and Gly in the linker is,respectively, from about 1:1 to about 1:10, from about 1:2 to about 1:6,or about 1:4. Exemplary linker sequences comprise S(GGS)₄GSS (SEQ ID NO:46), S(GGS)₉GS (SEQ ID NO: 47), (GGS)₉GS (SEQ ID NO: 48). In someembodiments, the linker has at least 10 amino acids, or at least 15amino acids, or at least 20 amino acids, or at least 25 amino acids. Forexample, the linker may have a length of from 15 to 30 amino acids. Invarious embodiments, longer linkers of at least 15 amino acids canprovide improvements in yield upon expression in Pichia pastoris.

In other embodiments, the linker is a physiologically-cleavable linker,such as a protease-cleavable linker. For example, the protease may be acoagulation pathway protease, such as activated Factor XII. In certainembodiments, the linker comprises the amino acid sequence of Factor XI(SEQ ID NO: 49) and/or prekallikrein (SEQ ID NO: 50 or 51) or aphysiologically cleavable fragment thereof. In other embodiments, thelinker includes a peptide sequence that is targeted for cleavage by aneutrophil specific protease, such as neutrophil elastase, cathepsin G,and proteinase 3.

In various embodiments, the human DNASE1-LIKE 1 (D1L2) variant evaluatedand selected for therapy in accordance with this disclosure comprises anamino acid sequence that is at least 80% identical to the enzyme definedby SEQ ID NO: 3, with one or more building block substitutions.

In some embodiments, the building block substitutions to D1L2 areselected from non-human D1L2 proteins and result in variants of humanD1L2, which feature one or more of the following mutations: L22K, I24V,I29V, S35N, S35H, S35R, S35T, V37A, S38L, A41D, A41V, A41G, G431, S44G,S44i, I45V, K48Q, L55I, L55V, A56T, A56M, P64A, 570D, 570T, A71T, A71L,A71S, A71V, M73L, E74Q, N77H, S78R, E81K, E81R, E83N, S85G, S85N, Q90E,Q90K, Q96H, F103Y, V104I, K107D, A109V, A109T, A109K, V110A, V113L,V113M, D114S, D114E, L117Q, P119S, E122G, V124A, V124F, S126N, E128D,F134V, A136V, A136T, G138S, G138R, T139S, T139C, S148C, A151P, P154A,A159P, A160G, A161P, A161T, Q162D, Q162K, Q162R, Q162T, N163K, N163E,L164V, L164F, I167V, H174N, Q175H, A178T, A178V, D192N, G195N, T196S,D198V, M199L, M199I, S210K, R213K, Q215H, A218P, A219S, E226Q, V227I,S243T, A252V, C253S, A255S, A255V, R256H, L257M, R259K, S260T, L261V,Q264H, T267S, T267A, D270N, G276D, G276S, T280S, T280D, T280A, A284C,I286V, L295F, F297S, F297T, F297P, H298R, and R299del.

In some embodiments, the building block substitutions to D1L2 areselected from human D1 and result in variants of human D1L2, whichfeature one or more of the following mutations: G2R, P4_A6delinsMK, A9G,W12L, EISA, A16_G18delinsLLQ, T19_A21delinsAVS, R23K, G25A, S31T,D34_S35delinsET, V37M, D39_G43delinsNATLV, I45Y, A47_K48delinsVQ,A51_G52delinsSR, L55I, P64_D65delinsSH, S67T, S70_A71delinsGK,M73_I76delinsLDNL, S78_E83delinsQDAPDT, S85_F86delinsHY, S88V, Q90E,D95_Q96delinsNS, M100R, K107P, A109Q, V112A, T115S, L117Y, P119D,P121_E122delinsGCEPCGN, V124T, S126N, F130_V131delinsI, K133R, S135G138delinsFSRF, G140_I167delinsEVREFAIV, H174_Q175delinsGD,I191_D192delinsQE, T196_D197delinsLE, M199_L202delinsVMLM, D208G,A214_D216delinsPSQ, A218_A219delinsSS, R223_S224delinsWT,E226_V227delinsPT, K229Q, V240_D244delinsATPTH, A252_C253delinsVA,A225_R256delinsML, R259_K262delinsGAVV, Q264D, T267_D270delinsLPFN,E273_E275delinsAAY, D278_Q281delinsSDQL, L283Q, F283Y, T294M, andF297_R299del.

In some embodiments, the building block substitutions to D1L2 areselected from human D1L1 and result in variants of human D1L2, whichfeature one or more of the following mutations: G2_G3delinsHY, RST,W12_E15delinsFLIL, A17N, T19_A20delinsAQ, L22F, G25C, I29A,S31_S35delinsRLTLA, S38_K48delinsAREQVMDTLVR, G52_Y53delinsRC,L55_V58delinsIMVL, R62V, P64_L66delins SSG, V69_A71delinsIPL,M73_I76delinsLREL, S78_E83delinsRFDGSGP, F86_V87delinsTL,Q90_P91delinsPQ, D95_Q96delinsST, K98M, M100T, L102_V104delinsVYF,K107_S111delinsSHKTQ, V113_T115delinsLSS, L117V, P119N,P121_E122delinsED, S126A, V132_K133delinsAQ, A136L, G138_P145delinsSNV,R149_N163del, I167V, A171_A172delinsTT, H174_A178delinsKAVEK,I180_D181delinsLN, Y187F, D189E, I191_K193delinsSQH, M199_F201delinsVIL,S210_R221delinsASLTKKRLDKLE, S224_V227delinsTEPG, K229H, L231V, P233A,S235_A235delinsGE, G241_D244delinsRASTH, A246T, I250V, A252_C253delinsLH, A255E, L257C, R259_S260delinsSL, K262_S265delinsHT,T267_H269delinsAAF, Q272_E274delinsPTS, G276Q, D278_Q281delinsTEEE,A284N, F289Y, T294E, and F297_R299delinsLSQAHSVQPLSTVLLLLSLLSPQLCPAA.

In some embodiments, the building block substitutions to D1L2 areselected from human DNASE1-LIKE 3 Isoform 1 (D1L3) and result invariants of human D1L2, which feature one or more of the followingmutations: G2_R5delinsSREL, L7P, A9_A10delinsL, W12_A13delinsLL,S15_A20delinsSIHSAL, L22M, G25_A26delinsCS, I29_Q30delinsVR, D34E,V37_S38delinsQE, P40_I45delinsKNAMDV, A47V, I49_Y53delinsVIKRS,L55_A56delinsII, Q59M, V61_R62delinsIK, P64_A71delinsSNNRICPI,Q75_I76delinsKL, N77_S78insRS, V79_E83delinsRRGIT, S85_F86delinsNY,S88I, Q90_P9ldelinsSR, D95_Q96delinsNT, M100Q, L102A, V104L,R106_A109delinsKEKL, V113_T115delinsKRS, L117H, P119H,P121_E122delinsYQDGDA, K133W, S135_A136delinsQS, G138H, G140_A159del,A161_L164delinsVKDF, L166I, A171_A172delinsTT, H174_A176delinsETS,A178K, A182E, Y184_D185delinsVE, L188T, I191_K193 delinsKHR,G195_L200delinsKAENFI, L202M, D208G, R213_D126delinsPKKA,A218_A129delinsKN, S224_V227delinsTDPR, K229V, P233G, S235_A236delinsQE,G241_N242delinsKK, S243_D244insTN, A252_C253 delinsLR,A255_R259delinsQEIVS, L261_K262delinsVV, Q264K, A266_T267delinsNS,H269F, E273_G276delinsKAYK, D278_Q281delinsTEEE, A284_I285delinDV,V293_T294delinsFK, K296_H298delinsQSS, and R295insAFTNSKKSVTLRKKTKSKRS.

In some embodiments, the building block substitutions to D1L2 areselected from human DNASE1-LIKE 3 Isoform 2 (D1L3-2) and result invariants of human D1L2, which feature one or more of the followingmutations: G2_R5delinsSREL, L7P, A9_A10delinsL, W12_A13delinsLL,S15_A20delinsSIHSAL, L22M, G25_A26delinsCS, I29_Q30delinsVR, D34E,V37_S38delinsQE, P40_I45delinsKNAMDV, A47V, I49_Y53delinsVIKRS,L55_A56delinsII, Q59M, V61_R62delinsIK, P64_A71delinsSNNRICPI,Q75_I76delinsKL, K107_A109delinsEKL, V113_T115delinsKRS, L117H, P119H,P121_E122delinsYQDGDA, K133W, S135_A136delinsQS, G138H, G140_A159del,A161_L164delinsVKDF, L166I, A171_A172delinsTT, H174_A176delinsETS,A178K, A182E, Y184_D185delinsVE, L188T, I191_K193delinsKHR,G195_L200delinsKAENFI, L202M, D208G, R213_D126delinsPKKA,A218_A129delinsKN, S224_V227delinsTDPR, K229V, P233G, S235_A236delinsQE,G241_N242delinsKK, S243_D244insTN, A252_C253delinsLR,A255_R259delinsQEIVS, L261_K262delinsVV, Q264K, A266_T267delinsNS,H269F, E273_G276delinsKAYK, D278_Q281delinsTEEE, A284_I285delinDV,V293_T294delinsFK, K296_H298delinsQSS, and R295insAFTNSKKSVTLRKKTKSKRS.

In certain embodiments, the D1L2 protein variant contains one or moreamino acid substitutions, additions, or deletions in the proline-richextension domain defined by SEQ ID NO: 9. The proline-rich extensiondomain or a portion thereof may be deleted, including a deletion (ortruncation) of at least 3 amino acids, at least 5 amino acids, or atleast 10 amino acids.

In various embodiments, the D1L2 variant evaluated in accordance withthe disclosure comprises the D1L2 wildtype amino acid sequence or avariant sequence described herein, fused to the C-terminus of a carrierprotein, with a linking amino acid sequence. In some embodiments, thecarrier protein is Fc fragment or albumin. In some embodiments, thecarrier protein is human albumin. The linking sequence can be a flexiblelinker predominately composed of Gly, or Gy and Ser. In someembodiments, the linker is composed of Gly and amino acids havinghydrophilic side chains (e.g., Ser, Thr). In some embodiments, thelinker is from 5 to 20 amino acids. An exemplary linker has thestructure (GGGGS)3.

The peptide linker may be a flexible linker, a rigid linker, or in someembodiments a physiologically-cleavable linker (e.g., aprotease-cleavable linker). In some embodiments, the linker is 5 to 100amino acids in length, or is 5 to 50 amino acids in length.

Linkers, where present, can be selected from flexible, rigid, andcleavable peptide linkers. Flexible linkers are predominately orentirely composed of small, non-polar or polar residues such as Gly, Serand Thr. An exemplary flexible linker comprises (Gly_(y)Ser)_(n)linkers, where y is from 1 to 10 (e.g., from 1 to 5), and n is from 1 toabout 10, and in some embodiments, is from 3 to about 6. In exemplaryembodiments, y is from 2 to 4, and n is from 3 to 8. Due to theirflexibility, these linkers are unstructured. More rigid linkers includepolyproline or poly Pro-Ala motifs and α-helical linkers. An exemplaryα-helical linker is A(EAAAK)_(n)A, where n is as defined above (e.g.,from 1 to 10, or 2 to 6). Generally, linkers can be predominatelycomposed of amino acids selected from Gly, Ser, Thr, Ala, and Pro.Exemplary linker sequences contain at least 10 amino acids, and may bein the range of 15 to 35 amino acids. Exemplary linker designs areprovided as SEQ ID NOS: 41 to 51.

In some embodiments, the variant comprises a linker, wherein the aminoacid sequence of the linker is predominately glycine and serineresidues, or consists essentially of glycine and serine residues. Insome embodiments, the ratio of Ser and Gly in the linker is,respectively, from about 1:1 to about 1:10, from about 1:2 to about 1:6,or about 1:4. Exemplary linker sequences comprise S(GGS)₄GSS (SEQ ID NO:46), S(GGS)₉GS (SEQ ID NO: 47), (GGS)₉GS (SEQ ID NO: 48). In someembodiments, the linker has at least 10 amino acids, or at least 15amino acids, or at least 20 amino acids, or at least 25 amino acids. Forexample, the linker may have a length of from 15 to 30 amino acids. Invarious embodiments, longer linkers of at least 15 amino acids canprovide improvements in yield upon expression in Pichia pastoris.

In other embodiments, the linker is a physiologically-cleavable linker,such as a protease-cleavable linker. For example, the protease may be acoagulation pathway protease, such as activated Factor XII. In certainembodiments, the linker comprises the amino acid sequence of Factor XI(SEQ ID NO: 49) and/or prekallikrein (SEQ ID NO: 50 or 51) or aphysiologically cleavable fragment thereof. In other embodiments, thelinker includes a peptide sequence that is targeted for cleavage by aneutrophil specific protease, such as neutrophil elastase, cathepsin G,and proteinase 3.

In some embodiments, the human DNASE1-LIKE 3 Isoform 1 (D1L3) variantevaluated and selected for use in therapy in accordance with embodimentsof the invention comprise an amino acid sequence that is at least 80%identical to the enzyme defined by SEQ ID NO: 4, with one or morebuilding block substitutions.

In some embodiments, the building block substitutions to D1L3 areselected from non-human D1L3 proteins and result in variants of humanD1L3 which feature one or more of the following mutations: M21L, K22R,I22L, I22V, E33A, E33Y, E33G, S34A, S34T, Q36K, Q36R, E37A, E37Q, D48N,K39Q, K39H, K39R, K39C, N40E, N40Q, N40K, A41V, V44I, V53I, I53L, I54M,V57L, 160V, N64S, H64S, R66N, R66M, 170V, 170M, 170T, M72L, E73K, K74R,R77G, R81K, G82S, I83V, I83T, T84M, T84K, S91P, T91V, T91A, L105V,K107M, V111L, S112T, R115T, R115A, R115K, R115D, R115Q, S116K, S116N,S116Y, H118L, H118V, Y119F, H120G, Y122N, Q123E, D124A, D124S, D124N,G125E, A127V, A127T, V129A, F135Y, V137T, Q140H, S141A, H143F, H143Y,V146A, I152V, T157S, T160A, V162I, K163R, V169A, E170D, T173M, T173L,V175M, K176R, K176Q, H177S, H177R, R178Q, K180E, K180N, K181T, K181V,E183A, E183Q, A201S, K203Q, K203R, R212K, R212N, R212G, R212M, V214I,G218K, G218A, Q220E, Q220D, K227R, K227S, K227E, N239K, N239S, N239H,R239C, Q241P, E242D, E242N, V244I, S245N, S245R, K250R, K250D, K250R,K250G, K250N, K250Q, N252S, S253G, S253L, V254T, V254I, D256N, Q258R,Y261F, K262D, K262E, K262L, K262R, K262Q, T264S, E266S, E267K, E267Q,E267K, D270N, D270E, V271I, S282E, R285T, F287I, S290N, K291R, V294I,T295S, T295Q, L296V, L296P, L296S, R297K, K299R, T300K, T300A, S302G,S302A, S302V, S302I, K303N, K303S, K303R, R304H, R304S, S305P, S305T,and S305A.

In some embodiments, the building block substitutions to D1L3 areselected from human D1 and result in variants of human D1L3 whichfeature one or more of the following mutations: M1_E4delinsMRGMKL,A6_P7delinsGA, L10A, L12_S17delinsAALLQG, L19_R22delinsVSLK,C24_S25delinsAA, V28_530delinsIQT, S34T, Q36_V44delinsMSNATLVSY,K47_K50delinsQILS, C52Y, I55A, M58Q, I60_K61delinsVR,N64_I70delinsHLTAVGK, M72_K74delinsLDN, R77_I83delinsQDAPD, N86H, I89V,S91_R92delinsEP, T97S, Q101R, A103L, L105V, K107_L110delinsRPDQ,V113_R115delinsAVD, H118Y, H120D, Y122_A127delinsGCEPCGN, V129T, S131N,F135_V136delinsAI, W138R, Q140F, P142_H143delinsRF, A145E,K147_D148delinsRE, V150A, I152V, I156_T157delinsAA, E159_S161delinsGDA,K163A, E167A, V169_E170delinsYD, T173L, K176_R178delinsQEK,K180_A181delinsGL, N183_F186delinsDVML, P198_A201delinsRPSQ,K203_N204delins55, R208W, D210S, R212T, V214Q, G218P, Q220_E221delinsSA,V225_S228delinsATP, N230H, L238_R239delinsVA, Q241_S246delinsMLLRGA,K250D, N252_V254delinsALP, D256N, K259A, K262G, T264_E267delinsSDQL,L269_V271delinsQAI, F275Y, F279_K280delinsVM, and Q282_S205delinsK.

In some embodiments, the building block substitutions to D1L3 areselected from human D1L1 and result in variants of human D1L3 whichfeature one or more of the following mutations: S2_L5delinsHYPT,P7_L9delinsL, L11F, L13_S17delinsILANG, L19delinsQ, M21F, S25A,V28_S34delinsAQRLTLA, Q36_A41delinsVAREQV, V44_I45delinsTL,K47_K50delinsRILA, I55_M58delinsMVLQ, I60_K61delinsVV,N6_C68delinsSGSAI, 170L, M72_L74delinsLRE, N78_R81delinsFDGS,I83_T84delinsP, N86_I89delinsSTLS, S91_R92delinsPQ, N96S, K99M, Q101T,A103_L105delinsVYF, K107_S112delinsRSHKTQ, K114_R115delinsLS, H118V,H120N, Y122_A127delinsED, S131A, V137_W138delinsAQ, Q140_S141delinsSL,H143_F149delinsSNVLPSL, I151_I152delinsLV, E159_V162delinsKAVE,I165_A167delinsLNA, V169_E170delinsYD, Y172_D174delinsFLE,K176_R178delinsSQH, K180_F184delinsQSKDV, G193D, S195_P198delinsASLT,A201_R206delinsRLDKLE, D210E, R212G, V214H, L216V, G218A, Q220G,K226_K227delinsRA, N230H, A232T, I236V, R239H, Q246_V249delinsERCR,S246_V254delinsLLHTAAA, Q258_K262delinsPTSFQ, D270_V271delinsNI, F275Y,F279_K280delinsVE, Q282_S283delinsKL, R285Q, F287_K292delinsHSVQPL,V294L, and L296_S205delinsVLLLLSLLSPQLCPAA.

In some embodiments, the building block substitutions to D1L3 areselected from human D1L2 and result in variants of human D1L3 whichfeature one or more of the following mutations: S2_L5delinsGGPR, P7L,L9delinsAA, L11_L12delinsWA, S14_L19delinsEAAGTA, M21L, C24_S25delinsGA,V28_R29delinsIQ, E33D, Q36_E37delinsVS, K39_V44delinsPACGSI, A47V,V48_C52delinsILAGY, I54_I55delinsLA, M58Q, I60_K61delinsVR,S63_I70delinsPDLSAVSA, K74_L75delinsQI, XX (deletion in Donor),R80_T84delinsVSEHE, N86_Y87delinsSF, 189S, S91_R92delinsQP,N96_T97delinsDQ, Q101M, A103L, L105V, K107_L110delinsRKDA,K114_S116delinsVDT, H118L, H120P, Y122_A127delinsPE, W138K,Q140_S141delinsSA, H143G, T144_A145insGERAPPLPSRRALTPPPLPA,V146_F149delinsAQNL, I151L, T156_T157delinsAA, E159_S161delinsHQA,K163A, E162A, V169_E170delinsYD, T173L, K176_R178delinsIDK,K180_I185delinsGTDDML, M187L, G193D, P198_A201delinsRAQD,K203_N204delinsAA, T209_R212delinsSSEV, V214K, G218P, Q220_E221delinsSA,K226_K227delinsGN, T229_N230delinsD, L238_R239delinsAC,Q241_S245delinsARLRR, V247_V248delinsLK, K250Q, N252_S253delinsAT,F255H, K259_K262delinsEEFG, T264_E267delinsDQTQ, D270_V271delinsAI,F279_K280delinsVT, Q282_S284delinsKFH, and A286_S305del.

In certain embodiments, the D1L3 protein variant contains one or moreamino acid substitutions, additions, or deletions in the C-terminal tailamino acid sequence defined by SEQ ID NO: 10. The C-terminal tail domainor a portion thereof may be deleted. In some embodiments, at least 3, 5,8, 10, 12, 15, 18, or 23 amino acids of the C-terminal tail domain aredeleted.

In certain embodiments, the D1L3 protein variant contains one or more,e.g., 1, 2, 3, 4, 5, or more amino acid substitutions, additions, ordeletions in the internal sequence defined by SEQ ID NO: 11 (which isabsent from isoform 2), and which is optionally deleted in whole or inpart.

In various embodiments, the D1L3 variant evaluated in accordance withthe disclosure comprises the D1L3 wildtype amino acid sequence or avariant sequence described herein, fused to the C-terminus of a carrierprotein, with a linking amino acid sequence. In some embodiments, thecarrier protein is Fc fragment or albumin. In some embodiments, thecarrier protein is human albumin. The linking sequence can be a flexiblelinker predominately composed of Gly, or Gy and Ser. In someembodiments, the linker is composed of Gly and amino acids havinghydrophilic side chains (e.g., Ser, Thr). In some embodiments, thelinker is from 5 to 20 amino acids. An exemplary linker has thestructure (GGGGS)₃.

The peptide linker may be a flexible linker, a rigid linker, or in someembodiments a physiologically-cleavable linker (e.g., aprotease-cleavable linker). In some embodiments, the linker is 5 to 100amino acids in length, or is 5 to 50 amino acids in length.

Linkers, where present, can be selected from flexible, rigid, andcleavable peptide linkers. Flexible linkers are predominately orentirely composed of small, non-polar or polar residues such as Gly, Serand Thr. An exemplary flexible linker comprises (Gly_(y)Ser)_(n)linkers, where y is from 1 to 10 (e.g., from 1 to 5), and n is from 1 toabout 10, and in some embodiments, is from 3 to about 6. In exemplaryembodiments, y is from 2 to 4, and n is from 3 to 8. Due to theirflexibility, these linkers are unstructured. More rigid linkers includepolyproline or poly Pro-Ala motifs and α-helical linkers. An exemplaryα-helical linker is A(EAAAK)_(n)A, where n is as defined above (e.g.,from 1 to 10, or 2 to 6). Generally, linkers can be predominatelycomposed of amino acids selected from Gly, Ser, Thr, Ala, and Pro.Exemplary linker sequences contain at least 10 amino acids, and may bein the range of 15 to 35 amino acids. Exemplary linker designs areprovided as SEQ ID NOS: 41 to 51.

In some embodiments, the variant comprises a linker, wherein the aminoacid sequence of the linker is predominately glycine and serineresidues, or consists essentially of glycine and serine residues. Insome embodiments, the ratio of Ser and Gly in the linker is,respectively, from about 1:1 to about 1:10, from about 1:2 to about 1:6,or about 1:4. Exemplary linker sequences comprise S(GGS)₄GSS (SEQ ID NO:46), S(GGS)₉GS (SEQ ID NO: 47), (GGS)₉GS (SEQ ID NO: 48). In someembodiments, the linker has at least 10 amino acids, or at least 15amino acids, or at least 20 amino acids, or at least 25 amino acids. Forexample, the linker may have a length of from 15 to 30 amino acids. Invarious embodiments, longer linkers of at least 15 amino acids canprovide improvements in yield upon expression in Pichia pastoris.Further, longer linker sequences showed improved chromatin-degradingactivity, as compared to shorter linker sequences.

In other embodiments, the linker is a physiologically-cleavable linker,such as a protease-cleavable linker. For example, the protease may be acoagulation pathway protease, such as activated Factor XII. In certainembodiments, the linker comprises the amino acid sequence of Factor XI(SEQ ID NO: 49) and/or prekallikrein (SEQ ID NO: 50 or 51) or aphysiologically cleavable fragment thereof. In other embodiments, thelinker includes a peptide sequence that is targeted for cleavage by aneutrophil specific protease, such as neutrophil elastase, cathepsin G,and proteinase 3.

In some embodiments, the DNASE1-LIKE 3 Isoform 2 (D1L3-2) variantevaluated and selected for use in therapy in accordance with embodimentsof the invention comprise an amino acid sequence that is at least 80%identical to the enzyme defined by SEQ ID NO: 5, with one or morebuilding block substitutions.

In some embodiments, the building block substitutions to D1L3-2 areselected from non-human D1L3 proteins and result in variants of humanD1L3-2 which feature one or more of the following mutations: M21L, K22R,I22L, I22V, E33A, E33Y, E33G, S34A, S34T, Q36K, Q36R, E37A, E37Q, D48N,K39Q, K39H, K39R, K39C, N40E, N40Q, N40K, A41V, V44I, V53I, I53L, I54M,V57L, 160V, N64S, H64S, R66N, R66M, 170V, 170M, 170T, M72L, E73K, K74R,R77G, V81L, S82T, R85T, R85A, R85K, R85D, R85Q, S86K, S86N, S86Y, H88L,H88V, Y89F, H90G, Y92N, Q93E, D94A, D94S, D94N, G95E, A97V, A97T, V99A,F105Y, V107T, Q110H, S111A, H113F, H113Y, V116A, I122V, T127S, T130A,V132I, K133R, V139A, E140D, T143M, T143L, V145M, K146R, K146Q, H147S,H147R, R148Q, K150E, K150N, K151T, K151V, E153A, E153Q, A171S, K173Q,K173R, R182K, R182N, R182G, R182M, V184I, G188K, G188A, Q190E, Q190D,K197R, K197S, K197E, N209K, N209S, N209H, R209C, Q211P, E212D, E212N,V214I, S215N, S215R, K220R, K220D, K220R, K220G, K220N, K220Q, N222S,S223G, S223L, V224T, V224I, D226N, Q228R, Y231F, K232D, K232E, K232L,K232R, K232Q, T234S, E236S, E237K, E237Q, E237K, D240N, D240E, V241I,S252E, R255T, F257I, S260N, K261R, V264I, T265S, T265Q, L266V, L266P,L266S, R267K, K269R, T270K, T270A, S272G, S272A, S272V, S272T, K273N,K273S, K273R, R274H, R274S, S275P, S275T, and S275A.

In some embodiments, the building block substitutions to D1L3-2 areselected from human D1 and result in variants of human D1L3-2 whichfeature one or more of the following mutations: M1_E4delinsMRGMKLL,A6_P7delinsGA, L10A, L12_S17delinsAALLQG, L19_R22delinsVSLK,C24_S25delinsAA, V28_S30delinsIQT, S34T, Q36_V44delinsMSNATLVSY,K152_K156delinsGDAVA, C52Y, I55A, M58Q, I60_K61delinsVR,N64_I70delinsHLTAVGK, M72_K74delinsLDN,N76_R77insQDAPDTYHYVVSEPLGRNSYKERYLFVY, E78_L80delinsPDQ,V83_R85delinsAVD, H88Y, H90D, Y92_A97delinsGCEPCGN, V99T, S101N,F105_V106delinsAI, W108R, Q110F, P112_H113delinsRF, A115E,K117_D118delinsRE, V120A, I122V, T126_T127delinsAA, E129_T131delinsGDA,K133A, V139_E140delinsYD, T143L, K146_R148delinsQEK, K150_A151delinsGL,N153_F156delinsDVML, P168_A171delinsRPSQ, K173_N1174delinsSS, R178W,D180S, R182T, V184Q, G188P, Q190_E191delinsSA, V195_S199delinsATP,N200H, L208_R209delinsVA, Q211_S216delinsMLLRGA, K220D,N222_V224delinsALP, D226N, K232G, T234_E237delinsSDQL,L239_V241delinsQAI, F245Y, F249_K250delinsVM, and Q252_S275delinsK.

In some embodiments, the building block substitutions to D1L3-2 areselected from human D1L1 and result in variants of human D1L3-2 whichfeature one or more of the following mutations: S2_L5delinsHYPT,P7_L9delinsA, L11F, L13_S17delinsILANG, L19delinsQ, M21F, S25A,V28_S34delinsAQRLTLA, Q36_A41delinsVAREQV, V44_I45delinsTL,K47_K50delinsRILA, I55_M58delinsMVLQ, I60_K61delinsVV,N6_C68delinsSGSAI, 170L, M72_L74delinsLRE,N76_R77insRFDGSGPYSTLSSPQLGRSTYMETYVYFYRSHKTQ, E78_S82delinsSHKTQ,K84_R85delinsLS, H88V, H90N, Y92_A97delinsED, S101A, V107_W108delinsAQ,Q110_S111delinsSL, H113_F119delinsSNVLPSL, I121_I122delinsLV,E129_V132delinsKAVE, I135_A137delinsLNA, V139_E140delinsYD,Y142_D144delinsFLE, K146_R148delinsSQH, K150_F154delinsQSKDV, G163D,S165_P168delinsASLT, A171_R176delinsRLDKLE, D180E, R182G, V184H, L186V,G188A, Q190G, K196_K227delinsRA, N200H, A192T, I206V, R209H,Q216_V219delinsERCR, S216_V224delinsLLHTAAA, Q228_K232delinsPTSFQ,D240_V241delinsNI, F245Y, F249_K250delinsVE, Q252_S253delinsKL, R255Q,F257_K262delinsHSVQPL, V264L, and L266_S275delinsVLLLLSLLSPQLCPAA.

In some embodiments, the building block substitutions to D1L3-2 areselected from human D1L2 and result in variants of human D1L3-2 whichfeature one or more of the following mutations: S2_L5delinsGGPR, P7L,L9delinsAA, LL11_L12delinsWA, S14_L19delinsEAAGTA, M21L,C24_S25delinsGA, V28_R29delinsIQ, E33D, Q36_E37delinsVS,K39_V44delinsPACGSI, A47V, V48_C52delinsILAGY, I54_I55delinsLA, M58Q,I60_K61delinsVR, S63_I70delinsPDLSAVSA, K74_L75delinsQI,E78_L80delinsKDA, K84_S86delinsVDT, H88L, H90P, Y92_A97delinsPE, W108K,Q110_S111delinsSA, H113G, T114_A115insGERAPPLPSRRALTPPPLPA,V116_F119delinsAQNL, I121L, T126_T127delinsAA, E129_S141delinsHQA,K133A, E132A, V139_E140delinsYD, T143L, K146_R148delinsIDK,K150_I155delinsGTDDML, M157L, G163D, P168_A171 delinsRAQD,K173_N174delinsAA, T179_R182delinsSSEV, V184K, G188P, Q190_E191delinsSA,K196_K197delinsGN, T199_N200delinsD, L208_R209delinsAC,Q211_S215delinsARLRR, V217_V218delinsLK, K220Q, N222_S223delinsAT,F225H, K229_K242delinsEEFG, T234_E237delinsDQTQ, D240_V241delinsAI,F249_K250delinsVT, and Q252_S254delinsKFH, A256_S275del.

In certain embodiments, the D1L3-2 protein variant contains one or moreamino acid substitutions, additions, or deletions in the C-terminal taildomain defined by SEQ ID NO: 11. The C-terminal tail domain or a portionthereof may be deleted. In some embodiments, at least 3, 5, 8, 10, 12,15, 18, or 23 amino acids of the C-terminal tail domain are deleted.

In various embodiments, the D1L3-2 variant evaluated in accordance withthe disclosure comprises the D1L3-2 wildtype amino acid sequence or avariant sequence described herein, fused to the C-terminus of a carrierprotein, with a linking amino acid sequence. In some embodiments, thecarrier protein is Fc fragment or albumin. In some embodiments, thecarrier protein is human albumin. The linking sequence can be a flexiblelinker predominately composed of Gly, or Gy and Ser. In someembodiments, the linker is composed of Gly and amino acids havinghydrophilic side chains (e.g., Ser, Thr). In some embodiments, thelinker is from 5 to 20 amino acids. An exemplary linker has thestructure (GGGGS)₃.

The peptide linker may be a flexible linker, a rigid linker, or in someembodiments a physiologically-cleavable linker (e.g., aprotease-cleavable linker). In some embodiments, the linker is 5 to 100amino acids in length, or is 5 to 50 amino acids in length.

Linkers, where present, can be selected from flexible, rigid, andcleavable peptide linkers. Flexible linkers are predominately orentirely composed of small, non-polar or polar residues such as Gly, Serand Thr. An exemplary flexible linker comprises (Gly_(y)Ser)_(n)linkers, where y is from 1 to 10 (e.g., from 1 to 5), and n is from 1 toabout 10, and in some embodiments, is from 3 to about 6. In exemplaryembodiments, y is from 2 to 4, and n is from 3 to 8. Due to theirflexibility, these linkers are unstructured. More rigid linkers includepolyproline or poly Pro-Ala motifs and α-helical linkers. An exemplaryα-helical linker is A(EAAAK)_(n)A, where n is as defined above (e.g.,from 1 to 10, or 2 to 6). Generally, linkers can be predominatelycomposed of amino acids selected from Gly, Ser, Thr, Ala, and Pro.Exemplary linker sequences contain at least 10 amino acids, and may bein the range of 15 to 35 amino acids. Exemplary linker designs areprovided as SEQ ID NOS: 41 to 51.

In some embodiments, the variant comprises a linker, wherein the aminoacid sequence of the linker is predominately glycine and serineresidues, or consists essentially of glycine and serine residues. Insome embodiments, the ratio of Ser and Gly in the linker is,respectively, from about 1:1 to about 1:10, from about 1:2 to about 1:6,or about 1:4. Exemplary linker sequences comprise S(GGS)₄GSS (SEQ ID NO:46), S(GGS)₉GS (SEQ ID NO: 47), (GGS)₉GS (SEQ ID NO: 48). In someembodiments, the linker has at least 10 amino acids, or at least 15amino acids, or at least 20 amino acids, or at least 25 amino acids. Forexample, the linker may have a length of from 15 to 30 amino acids. Invarious embodiments, longer linkers of at least 15 amino acids canprovide improvements in yield upon expression in Pichia pastoris, andmay provide for improved chromatin degrading activity.

In other embodiments, the linker is a physiologically-cleavable linker,such as a protease-cleavable linker. For example, the protease may be acoagulation pathway protease, such as activated Factor XII. In certainembodiments, the linker comprises the amino acid sequence of Factor XI(SEQ ID NO: 49) and/or prekallikrein (SEQ ID NO: 50 or 51) or aphysiologically cleavable fragment thereof. In other embodiments, thelinker includes a peptide sequence that is targeted for cleavage by aneutrophil specific protease, such as neutrophil elastase, cathepsin G,and proteinase 3.

In various embodiments, the DNASE2A (D2A) variant evaluated and selectedfor use in therapy in accordance with embodiments of the inventioncomprise an amino acid sequence that is at least 80% identical to theenzyme defined by SEQ ID NO: 6, with one or more building blocksubstitutions.

In some embodiments, the building block substitutions to DNASE2A areselected from non-human D2A proteins and result in variants of humanD2A, which feature one or more of the following mutations: Q25R, L38H,L38N, R39S, R39T, G40S, G42R, E43D, A44T, A44K, A44V, A45P, A45T, R47K,R47N, R47S, Q50T, Q50M, Q50R, L54M, L54F, E56Q, S57N, S57H, S57E, G59D,G59E, G60D, R62Q, R62S, R65V, R65A, A66G, L67Y, L67H, L67F, L67S, N69D,P71S, P71K, P71T, E72D, E72T, V75L, R77L, Q80L, R84Q, S85K, S85N, T87S,T87N, L93V, Q101K, P102S, P102Y, S103R, K104S, K104E, K104G, A105S,Q106R, Q106K, D107H, S109T, M110G, M110S, M110N, R111H, H122Q, D123E,V129I, N134R, P137S, P138R, A139S, A142G, A143V, S145T, H148P, S149N,S149G, C151Q, C151R, T152K, Y153F, L158I, F162L, F164L, A165T, A165S,S168A, S168P, S168L, K169R, K169G, K169D, K169N, M170I, G171S, K172R,W180L, W180M, N183D, Y184H, Q185K, Q185R, I180F, I180D, Q192R, E193K,F194L, D196Y, N199T, N199E, V201I, V201T, G203N, G203Q, S207L, S207R,Q208H, Q208R, E209G, I215V, T216I, Q220R, Q220K, A221K, A223T, V224T,V224S, F231C, S232G, K233N, A244S, A245E, T249S, N250T, H257Q, H257P,T259S, V260P, V260S, V260A, D269G, I270A, I270T, I270V, W271Y, W271H,W271Q, Q272K, Q272H, V273I, L274F, N275D, N277T, Q278E, 12791, A280G,A285S, G286R, P287L, S288T, S288A, S288N, N290S, S291A, S301A, S301T,K303Q, K303E, G304R, I307A, I307V, Q316K, G317A, G317R, E319T, Q320H,L326V, A328T, L330V, L330M, A332S, L333F, Q338R, Q338K, P339S, N343D,N343A, Y344W, Y344C, Q345K, and Q345E.

In some embodiments, the building block substitutions to D2A areselected from human DNASE2B (D2B) and result in variants of human D2A,which feature one or more of the following mutations: 12 L6delinsKQKMM,A8R, C11_P13delinsRTSFALLFLGLFGVLG, G15 T18delinsATIS, Y20S23delinsRNEE, Q25 P26delinsKA, V31_V32delinsTF, A37 L38delinsK, G40G42delinsQNK, A44_R47delinsSGET, Q50E, K52L, E56 G60delinsSTIRS,D62_A66delinsKSEQ, I68M, S70_V75delinsDTKSVL, S78T, P81Q, R84delinsEAYA,N86 L90delinsKSNNT, F92Y, L941, Q108 P109delinsGV, Q101K, S103D107delinsVNK, V117L, L120 G124delinsWNRVQ, V129I, V132I, N134Q,P136del, A139_A143delinsIPEEG, S145 W146delinsDY, H148 Y153delinsPTGRRN,T156_L158delinsSGI, V160 S161 delinsIT, P163_A165delinsKYN, F167K172delinsYEAIDS, T175 Y178delinsLVCN, W180N, N183 I89delinsSCSIPAT,A192H, F194_V194delinsLIHMPQLCTRASS, Q208 E209delinsEI, W211I215delinsGRLLT, T218Q, Q220_A221delnsAQ, A223_V224delinsQK,Q226_S227delinsLH, F231_K233delinsSDS, G235L, L238_G241delinsIFAA,L243M, A245_A246delinsQR, G248K, N250H, Q252_F255delinsLTET,H257_I262delinsQRKRQE, D269 Q272delinsLPYH, L274Y, Y276 Q278delinsIKA,A280 S288delinsKLSRHSY, N290S, T292_E293delinsYQ, S296A, V300I, P302Q,P305delinsTKNR, V309I, M312L, N315 Q320deinsSPHQAF, G322S,T325_L326delinsFI, L330 K335delinsNWQIYQ, P339G, K342_N343delinsLY,Q345_P346delinsES, and N348 I360delinsK.

In various embodiments, the D2A variant evaluated in accordance with thedisclosure comprises the D2A wildtype amino acid sequence or a variantsequence described herein, fused to the C-terminus of a carrier protein,with a linking amino acid sequence. In some embodiments, the carrierprotein is Fc fragment or albumin. In some embodiments, the carrierprotein is human albumin. The linking sequence can be a flexible linkerpredominately composed of Gly, or Gy and Ser. In some embodiments, thelinker is composed of Gly and amino acids having hydrophilic side chains(e.g., Ser, Thr). In some embodiments, the linker is from 5 to 20 aminoacids. An exemplary linker has the structure (GGGGS)₃.

The peptide linker may be a flexible linker, a rigid linker, or in someembodiments a physiologically-cleavable linker (e.g., aprotease-cleavable linker). In some embodiments, the linker is 5 to 100amino acids in length, or is 5 to 50 amino acids in length.

Linkers, where present, can be selected from flexible, rigid, andcleavable peptide linkers. Flexible linkers are predominately orentirely composed of small, non-polar or polar residues such as Gly, Serand Thr. An exemplary flexible linker comprises (Gly_(y)Ser)_(n)linkers, where y is from 1 to 10 (e.g., from 1 to 5), and n is from 1 toabout 10, and in some embodiments, is from 3 to about 6. In exemplaryembodiments, y is from 2 to 4, and n is from 3 to 8. Due to theirflexibility, these linkers are unstructured. More rigid linkers includepolyproline or poly Pro-Ala motifs and α-helical linkers. An exemplaryα-helical linker is A(EAAAK)_(n)A, where n is as defined above (e.g.,from 1 to 10, or 2 to 6). Generally, linkers can be predominatelycomposed of amino acids selected from Gly, Ser, Thr, Ala, and Pro.Exemplary linker sequences contain at least 10 amino acids, and may bein the range of 15 to 35 amino acids. Exemplary linker designs areprovided as SEQ ID NOS: 41 to 51.

In some embodiments, the variant comprises a linker, wherein the aminoacid sequence of the linker is predominately glycine and serineresidues, or consists essentially of glycine and serine residues. Insome embodiments, the ratio of Ser and Gly in the linker is,respectively, from about 1:1 to about 1:10, from about 1:2 to about 1:6,or about 1:4. Exemplary linker sequences comprise S(GGS)₄GSS (SEQ ID NO:46), S(GGS)₉GS (SEQ ID NO: 47), (GGS)₉GS (SEQ ID NO: 48). In someembodiments, the linker has at least 10 amino acids, or at least 15amino acids, or at least 20 amino acids, or at least 25 amino acids. Forexample, the linker may have a length of from 15 to 30 amino acids. Invarious embodiments, longer linkers of at least 15 amino acids canprovide improvements in yield upon expression in Pichia pastoris.

In other embodiments, the linker is a physiologically-cleavable linker,such as a protease-cleavable linker. For example, the protease may be acoagulation pathway protease, such as activated Factor XII. In certainembodiments, the linker comprises the amino acid sequence of Factor XI(SEQ ID NO: 49) and/or prekallikrein (SEQ ID NO: 50 or 51) or aphysiologically cleavable fragment thereof. In other embodiments, thelinker includes a peptide sequence that is targeted for cleavage by aneutrophil specific protease, such as neutrophil elastase, cathepsin G,and proteinase 3.

In some embodiments, the DNASE2B (D2B) variant evaluated and selectedfor therapy in accordance with embodiments of this disclosure comprisean amino acid sequence that is at least 80% identical to the enzymedefined by SEQ ID NO: 7, with one or more building block substitutions.

In some embodiments, the building block substitutions to D2B areselected from non-human D2B proteins and result in variants of humanD2B, which feature one or more of the following mutations: A28P, A28T,T29E, T29V, T29K, S31A, R33I, N34S, E36Y, E36D, A37P, T44I, T44A, T44V,K50R, R51Q, R51K, Q52T, N53S, N53D, N53E, K54R, E55A, E55G, S56G, G57E,G57T, G57R, T59A, T59M, E62Q, E62D, E62G, T70R, T70M, T701, R71Q, S72T,R74N, R74S, R74K, K75R, E77L, E77H, E77K, Q78Y, Q78H, Q78L, M80I, M80V,D82T, D82S, D82A, T83S, K84R, K84D, V86A, V86S, Q92E, Q93H, E96D, A97T,Y98H, Y98N, Y98C, A99D, A99H, S100A, S100F, K101E, S102T, S102N, S102D,N104D, N104S, L108V, I109L, G113A, V114I, K116G, K116A, P117S, V118A,N119T, N119G, N119S, Y120C, R122G, K123Q, K123N, Y124F, T127A, L132V,V136T, V136I, I145V, Q147K, Q147R, I151V, I151T, E154H, E154K, D157E,P160T, P160S, T161S, R164Q, N165Y, N165H, G166A, S168T, S168A, S168N,I170L, I170M, F174L, K175G, K175R, N178S, Y179F, A181E, A181T, S184F,V188I, C189L, C189F, C189Y, N190Q, V192I, S195R, S197F, A200S, A200N,A200T, T201I, T201A, H203R, Q204W, Q204M, E205K, I207V, I207F, H208Y,H208Y, M209L, Q211R, L212M, T214A, R215K, R215G, A216S, S217T, S217H,S218A, S219L, E220K, G223V, G223S, R224Q, L225Y, L225R, L225H, T227A,T228E, T228V, T228S, Q230H, Q233R, Q235L, K236N, K236S, L238V, L238I,S243F, D244S, D244T, S245F, F246Y, L247T, L247H, A252T, A252V, A253G,M255I, R258K, R258H, R258Q, T261V, T265A, T265V, E266Q, T267S, R270K,R272K, R272N, R272G, Q273H, Y283H, C285I, I288V, A290S, K292G, K292R,L293V, L293G, L293I, R295G, R295S, R295L, R295H, H296K, H296Q, Y298D,S300P, Y302R, Y302H, Q303H, A306S, I310V, Q312I, Q312T, Q312R, Q312L,G314D, G314R, T315S, K316A, K316Q, N317A, R318H, P329L, H330Y, F333L,F333S, S335G, T341S, T341N, Q342K, W344H, W344R, W344Q, Q345H, Q345Y,Q345R, Q345N, Q349H, Q351H, Q351D, Q351E, G352K, G352R, V354Y, L355S,Y356R, Y356H, Y357H, E358G, E358A, S359F, S359N, S359D, and K361N.

In others embodiments, the building block substitutions to D2B areselected from human DNASE2A (D2A) and result in variants of human D2B,which feature one or more of the following mutations: K2_M6delinsIPLLL,R8A, R11_G21delinsCVP, A28_53I delinsGALT, R33_E36delinsYGDS,K38_A39delinsQP, T44_F45delinsVV, K50delinsAL, Q52_K54delinsGSG,S56_T59delinsAAQR, E62Q, L64K, S70_S72delinsESSGG, K75_Q78delinsDGRA,M80I, D82_K87delinsSPEGAV, T905, Q93P, E96_Y99delinsR,K101_T105delinsNTSQL, Y107F, I109L, G113_V114delinsQP, K106Q,V118_Y120delinsSKAQD, L130V, W133_Q137delinsLDHDG, I142V, I145V, Q147N,F148_P149insP, I151_G155delinsASSAA, D157_Y158delinsSW,P160_N165delinsHSACTY, S168_I170delinsTLL, I172_T173delinsVS,K175_N177delinsPFA, Y179_s184delinsFSKMGK, L187_N190delinsTYTY, N192W,S195_T201delinsNYQLEGI, H203A, L206_S218delinsFPDLENVVKGHHV,E220_I221delinsQE, G223_T227delinsWNSSI, A232_Q233delinsQA,Q236_K236delinsAV, L238_H239delinsQ5, S243_S2456delinsFSK, L247G,I250_A253delinsLYSG, M255L, Q257_R258delinsAA, K260G, H262N,L264_T267delinsQVQF, Q269_E274delinsHKTVGI, L281_Y284delinsDIWQ, Y286L,I288_A290delinsVNQ, K292_Y298delinsAFPGAGPS, S300N, Y302_Q303delinsTE,A306S, I310V, Q312P, T315_R318delinsP, I322V, L325M,S328_F333delinsNQGEEQ, S335G, F338_I339delinsTL, N343_Q348delinsLPALWK,G353P, L355_Y356delinsKN, E358_S359delinsQP, andK361delinsNGMARKPSRAYKI.

In various embodiments, the D2B variant evaluated in accordance with thedisclosure comprises the D2B wildtype amino acid sequence or a variantsequence described herein, fused to the C-terminus of a carrier protein,with a linking amino acid sequence. In some embodiments, the carrierprotein is Fc fragment or albumin. In some embodiments, the carrierprotein is human albumin. The linking sequence can be a flexible linkerpredominately composed of Gly, or Gy and Ser. In some embodiments, thelinker is composed of Gly and amino acids having hydrophilic side chains(e.g., Ser, Thr). In some embodiments, the linker is from 5 to 20 aminoacids. An exemplary linker has the structure (GGGGS)₃.

The peptide linker may be a flexible linker, a rigid linker, or in someembodiments a physiologically-cleavable linker (e.g., aprotease-cleavable linker). In some embodiments, the linker is 5 to 100amino acids in length, or is 5 to 50 amino acids in length.

Linkers, where present, can be selected from flexible, rigid, andcleavable peptide linkers. Flexible linkers are predominately orentirely composed of small, non-polar or polar residues such as Gly, Serand Thr. An exemplary flexible linker comprises (Gly_(y)Ser)_(n)linkers, where y is from 1 to 10 (e.g., from 1 to 5), and n is from 1 toabout 10, and in some embodiments, is from 3 to about 6. In exemplaryembodiments, y is from 2 to 4, and n is from 3 to 8. Due to theirflexibility, these linkers are unstructured. More rigid linkers includepolyproline or poly Pro-Ala motifs and α-helical linkers. An exemplaryα-helical linker is A(EAAAK)_(n)A, where n is as defined above (e.g.,from 1 to 10, or 2 to 6). Generally, linkers can be predominatelycomposed of amino acids selected from Gly, Ser, Thr, Ala, and Pro.Exemplary linker sequences contain at least 10 amino acids, and may bein the range of 15 to 35 amino acids. Exemplary linker designs areprovided as SEQ ID NOS: 41 to 51.

In some embodiments, the variant comprises a linker, wherein the aminoacid sequence of the linker is predominately glycine and serineresidues, or consists essentially of glycine and serine residues. Insome embodiments, the ratio of Ser and Gly in the linker is,respectively, from about 1:1 to about 1:10, from about 1:2 to about 1:6,or about 1:4. Exemplary linker sequences comprise S(GGS)₄GSS (SEQ ID NO:46), S(GGS)₉GS (SEQ ID NO: 47), (GGS)₉GS (SEQ ID NO: 48). In someembodiments, the linker has at least 10 amino acids, or at least 15amino acids, or at least 20 amino acids, or at least 25 amino acids. Forexample, the linker may have a length of from 15 to 30 amino acids. Invarious embodiments, longer linkers of at least 15 amino acids canprovide improvements in yield upon expression in Pichia pastoris.

In other embodiments, the linker is a physiologically-cleavable linker,such as a protease-cleavable linker. For example, the protease may be acoagulation pathway protease, such as activated Factor XII. In certainembodiments, the linker comprises the amino acid sequence of Factor XI(SEQ ID NO: 49) and/or prekallikrein (SEQ ID NO: 50 or 51) or aphysiologically cleavable fragment thereof. In other embodiments, thelinker includes a peptide sequence that is targeted for cleavage by aneutrophil specific protease, such as neutrophil elastase, cathepsin G,and proteinase 3.

In some embodiments, the DNASE variant (e.g., a variant of DNASE1 (D1),DNASE1-LIKE 1 (D1L1), DNASE1-LIKE 2 (D1L2), DNASE1-LIKE 3 Isoform 1(D1L3), DNASE1-LIKE 3 Isoform 2 (D1L3-2), DNASE2A (D2A), and DNASE2B(D2B)) comprises an N-terminal or C-terminal fusion to a half-lifeextending moiety, such as albumin, transferrin, an Fc, or elastin-likeprotein. See U.S. Pat. No. 9,458,218, which is hereby incorporated byreference in its entirety. In some embodiments, the DNASE variant isdimerized by an immunoglobulin hinge region. For example, the engineeredenzymes described herein may also include an Fc-fusion domain (e.g. ahinge and CH2 domains and CH3 domains of an immunoglobulin). In othercases, the engineered DNASE variant is fused to albumin, e.g., humanalbumin (SEQ ID NO: 12) or a fragment thereof. See WO 2015/066550; U.S.Pat. No. 9,221,896, which are hereby incorporated by reference in itsentirety. Albumin can be fused at the N-terminus or the C-terminus ofthe engineered DNASE variant, and may optionally comprise an amino acidlinker. In some embodiments, two DNASE variants are dimerized by an Fchinge region, creating a dimeric molecule with synergistic functionalproperties for degrading NETs.

In some embodiments, human albumin and a flexible linker is fused to theN-terminus of DNASE1 (e.g., SEQ ID NO: 13), DNASE1-LIKE 1 (e.g., SEQ IDNO: 14), DNASE1-LIKE 2 (e.g., SEQ ID NO: 15), DNASE1-LIKE 3 Isoform 1(e.g., SEQ ID NO: 16), DNASE1-LIKE 3 Isoform 2 (e.g., SEQ ID NO: 17),DNASE2A (e.g., SEQ ID NO: 18), and DNASE2B (e.g., SEQ ID NO: 19).

In some embodiments, the recombinant DNASE variant comprises one or morepolyethylene glycol (PEG) moieties, which may be conjugated at one ormore of positions or the C-terminus. In some embodiments, the nativeamino acid at that position is substituted with an amino acid having aside chain suitable for crosslinking with hydrophilic moieties, tofacilitate linkage of the hydrophilic moiety to the peptide. In otherembodiments, an amino acid modified to comprise a hydrophilic group isadded to the peptide at the C-terminus. The PEG chain(s) may have amolecular weight in the range of about 500 to about 40,000 Daltons. Insome embodiments, the PEG chain(s) have a molecular weight in the rangeof about 500 to about 5,000 Daltons. In some embodiments, the PEGchain(s) have a molecular weight of about 10,000 to about 20,000Daltons.

The extracellular DNASE variants can be screened in assays for alteredproperties, including altered pH and temperature optimum, requirementfor divalent cations for enzymatic activity, mechanisms of enzymaticinhibition (e.g. salt, divalent cations, actin, heparin, proteases),substrate affinity and specificity (e.g. single-stranded DNA,double-stranded DNA, chromatin, NETs, plasmid DNA, mitochondrial DNA),localization upon secretion (e.g. membrane-bound, extracellular matrix),localization signals (e.g. nuclear localization signal, membraneanchor), glycosylation sites, disulfide-bonds and unpaired cysteines,compatibility with GMP-compliant in vitro expression systems (e.g.bacteria. yeast, mammalian cells), compatibility with carriers (e.g.PEGylation, Fc fragment, albumin), compatibility with GMP-compliantpurification methods (e.g. anion exchange resins, cation exchangeresins), toxicological profile, tissue penetration, pharmacokinetics andpharmacodynamics.

In some embodiments, the DNASE variants are evaluated using an in vitronucleic acid degradation assay, which can employ single ordouble-stranded DNA, plasmid DNA, mitochondrial DNA, NETs, or may employchromatin. In some embodiments, the assay is a NET-degrading assay. Thein vitro assay can be performed under different conditions includingvarying pH, temperature, divalent cations, and/or salt, to evaluate theenzyme characteristics for clinical applications. In some embodiments,enzyme activity is evaluated with fusion to carrier proteins such asalbumin or Fc, or with PEGylation.

In some embodiments, the DNASE variants are evaluated for theirexpression potential in prokaryotic and/or eukaryotic (includingmammalian and non-mammalian) expression systems, including their ease ofexpression, yield of recombinant enzyme, ability to be secreted asactive protein, the lack of inclusion bodies, the presence of andidentification of sites of glycosylation, and ease of purification withor without purification tags. In some embodiments, enzyme expression isevaluated with fusion to carrier proteins such as albumin or Fc. In someembodiments, DNASE variants are evaluated with substitution of anyunpaired Cysteines.

In some embodiments, the DNASE variants are evaluated for short termand/or long term stability (e.g., upon storage for several months at 4°C. and/or room temperature). Stability can be evaluated by formation ofaggregates, change of composition color, and/or enzyme activity.

In some embodiments, the DNASE variants are evaluated in animal models,including for immunogenic potential (e.g., presence of anti-DNASEvariant antibodies), half-life in circulation, protease resistance,bioavailability, and/or NET-degrading activity. In some embodiments,activity is evaluated in disease models. Exemplary animal models mayinclude rodent models (mouse, rat, rabbit) or primate models (e.g.,chimpanzee).

In some embodiments, at least one DNASE variant is evaluated in agenetically modified mouse deficient in D1 and D1L3 activity, the mousefurther having a heterologous expression of a G-CSF polynucleotide(e.g., in hepatocyte cells) or induction of a sustained endogenous G-CSFexpression (e.g., via repetitive administration of microbial compounds).This mouse model accumulates NETs and rapidly develops NET-relatedvascular occlusions. In these embodiments, the invention comprisesselecting DNASE enzyme that reduces the accumulation of NETs. Theselected enzyme is formulated (as described) for administration to ahuman patient. One skilled in the art recognizes standard methods forgenerating double knockout Dnase1^(−/−), Dnase113^(−/−) mice. Detaileddescriptions can be found in, for example, U.S. Application PublicationNo. US 2019/0350178 and PCT International Patent Publication No. WO2019/036719, the disclosure of which is incorporated herein by referencethe in its entirety.

The invention further provides pharmaceutical compositions comprisingextracellular DNASE variant as described herein, or optionally thepolynucleotide or the vector as described, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition may be formulated forany administration route, including topical, parenteral, or pulmonaryadministration. In various embodiments, the composition is formulatedfor intradermal, intramuscular, intraperitoneal, intraarticular,intravenous, subcutaneous, intraarterial, oral, sublingual, pulmonary,or transdermal administration.

In various embodiments, a selected DNASE variant is formulated with a“pharmaceutically acceptable carrier”, which includes any carrier thatdoes not interfere with the effectiveness of the biological activity andis not toxic to the patient to whom it is administered. Examples ofsuitable pharmaceutical carriers are well known in the art and includephosphate buffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc. Suchcarriers can be formulated by conventional methods and can beadministered to the subject at a suitable dose. Preferably, thecompositions are sterile. These compositions may also contain adjuvantssuch as preservative, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents.

In other aspects, the invention provides a method for treating a subjectin need of extracellular DNA degradation, extracellular chromatindegradation, extracellular trap (ET) degradation and/or neutrophilextracellular trap (NET) degradation. The method comprises administeringa therapeutically effective amount of the extracellular DNASE variant orcomposition described herein. Exemplary indications where a subject isin need of extracellular DNA or chromatin degradation (including ET orNET degradation) are disclosed in PCT/US18/47084, the disclosure ofwhich is appended to this application.

The invention is further described with reference to the followingnon-limiting examples.

EXAMPLES Example 1: The Approach Used for Engineering DNASE Variants forTherapeutic Applications

DNASE1 (D1) forms along with DNASE1-LIKE 1 (D1L1), DNASE1-LIKE 2 (D1L2)and DNASE1-LIKE 3 (D1L3), the DNASE1-protein family, a group ofhomologous secreted DNase enzymes. DNASE2A and DNASE2B form anadditional group of homologous DNase enzymes. DNASE1- and DNASE2-proteinfamily members are evolutionary conserved and expressed in variousspecies, including humans. In general, all extracellular DNASE enzymesprovide drug candidates for therapies of diseases that are associatedNETs. However, the physical, enzymatic, toxicological, andpharmacokinetic properties of these enzymes are not ideal for clinicalapplications.

An engineered D1 variant that is resistant to actin has been generated.Actin is an inhibitor of wild type D1. In brief, the 3D structure of theactin-DNASE1 complex was generated and actin binding sites in D1 wereidentified. Next, recombinant D1 variants with amino acids substitutionsin the actin binding sites were expressed and tested for theirsensitivity towards actin inhibition. The mutation A136F in SEQ ID NO: 1was identified to generate the best actin-resistant D1 variants. SeeUlmer et al., PNAS USA Vol. 93, pp 8225-8229 (1996).

Rats express a D1 variant that is naturally resistant to actininhibition due to mutations in actin binding sites. Furthermore, theenzymatic activity of human D1L2 and D1L3 is not inhibited by actin.Indeed, human D1L3 features an F139, which corresponds to A136 in humanD1 and likely causes the actin-resistance of D1L3.

Without being bound by theory, it was proposed that enzymatic propertiesthat are favorable for development of therapy with extracellular DNASEenzymes can be transferred to human extracellular DNASE enzymes fromextracellular DNASE enzymes expressed in other species (e.g. rat) orfrom other members of the same extracellular DNASE protein family (e.g.DNASE1-protein family comprised of DNASE1 (D1), DNASE1-LIKE 1 (D1L1),DNASE1-LIKE 2 (D1L2), DNASE1-LIKE 3 Isoform 1 (D1L3), DNASE1-LIKE 3Isoform 2 (D1L3-2), and DNASE1-protein family comprised of DNASE2A(D2A), and DNASE2B (D2B)).

A protein engineering technology, termed Building Block ProteinEngineering, can be applied to members of the DNASE1 and DNASE2 proteinfamily and an extracellular DNASE (e.g. D1, D1L1, D1L2, D1L3, D1L3-2,D2A, D2B). Building Block Protein Engineering is based on the followingsteps: providing a protein-protein alignment of donor and recipientDNASE enzymes; identifying variable amino acid(s) for transfer, thevariable amino acid(s) being flanked by one or more conserved aminoacids in the donor and recipient DNase enzymes; substituting thevariable amino acid(s) of the recipient DNase with the variable aminoacid(s) of the donor DNase to create a chimeric DNase; and recombinantlyproducing the chimeric DNase.

This approach can generate two distinct types of libraries with variantsof extracellular DNASE enzymes: a library based on phylogeneticvariation of a human extracellular DNASE, and a library that is based onvariation among DNASE-family members (FIG. 1).

For example, FIG. 2 shows the alignment of human D1L3, with D1L3 fromother species, including chimpanzee, baboon, mouse, rat, rabbit, dog,pig, guinea pig, cow, and elephant, and which identifies non-conservedamino acids (Building Blocks) that when transferred to human D1L3 resultin phylogenetic variants of human D1L3. These feature the followingmutations: M21L, K22R, I22L, I22V, E33A, E33Y, E33G, S34A, S34T, Q36K,Q36R, E37A, E37Q, D48N, K39Q, K39H, K39R, K39C, N40E, N40Q, N40K, A41V,V44I, V53I, I53L, I54M, V57L, 160V, N64S, H64S, R66N, R66M, 170V, 170M,170T, M72L, E73K, K74R, R77G, R81K, G82S, I83V, I83T, T84M, T84K, S91P,T91V, T91A, L105V, K107M, V111L, S112T, R115T, R115A, R115K, R115D,R115Q, S116K, S116N, S116Y, H118L, H118V, Y119F, H120G, Y122N, Q123E,D124A, D124S, D124N, G125E, A127V, A127T, V129A, F135Y, V137T, Q140H,S141A, H143F, H143Y, V146A, I152V, T157S, T160A, V162I, K163R, V169A,E170D, T173M, T173L, V175M, K176R, K176Q, H177S, H177R, R178Q, K180E,K180N, K181T, K181V, E183A, E183Q, A201S, K203Q, K203R, R212K, R212N,R212G, R212M, V214I, G218K, G218A, Q220E, Q220D, K227R, K227S, K227E,N239K, N239S, N239H, R239C, Q241P, E242D, E242N, V244I, S245N, S245R,K250R, K250D, K250R, K250G, K250N, K250Q, N252S, S253G, S253L, V254T,V254I, D256N, Q258R, Y261F, K262D, K262E, K262L, K262R, K262Q, T264S,E266S, E267K, E267Q, E267K, D270N, D270E, V271I, S282E, R285T, F287I,S290N, K291R, V294I, T295S, T295Q, L296V, L296P, L296S, R297K, K299R,T300K, T300A, S302G, S302A, S302V, S302T, K303N, K303S, K303R, R304H,R304S, S305P, S305T, and S305A.

FIG. 3 shows the alignment of human D1L3 with human D1L1, and identifiesnon-conserved amino acids (Building Blocks) that when transferred tohuman D1L3 result in variants of human D1L3. These feature the followingmutations: S2_L5delinsHYPT, P7_L9delinsL, L11F, L13_S17delinsILANG,L19delinsQ, M21F, S25A, V28_S34delinsAQRLTLA, Q36_A41delinsVAREQV,V44_I45delinsTL, K47_K50delinsRILA, I55_M58delinsMVLQ, I60_K61delinsVV,N6_C68delinsSGSAI, 170L, M72_L74delinsLRE, N78_R81delinsFDGS,I83_T84delinsP, N86_I89delinsSTLS, S91_R92delinsPQ, N96S, K99M, Q101T,A103_L105delinsVYF, K107_S112delinsRSHKTQ, K114_R115delinsL S, H118V,H120N, Y122_A127delinsED, S131A, V137_W138delinsAQ, Q140_S141delinsSL,H143_F149delinsSNVLPSL, I151_I152delinsLV, E159_V162delinsKAVE,I165_A167delinsLNA, V169_E170delinsYD, Y172_D174delinsFLE,K176_R178delinsSQH, K180_F184delinsQSKDV, G193D, S195_P198delinsASLT,A201_R206delinsRLDKLE, D210E, R212G, V214H, L216V, G218A, Q220G,K226_K227delinsRA, N230H, A232T, I236V, R239H, Q246_V249delinsERCR,S246_V254delinsLLHTAAA, Q258_K262delinsPTSFQ, D270_V271delinsNI, F275Y,F279_K280delinsVE, Q282_S283delinsKL, R285Q, F287_K292delinsHSVQPL,V294L, L296_S205delinsVLLLLSLLSPQLCPAA.

Such extracellular DNASE variants can be screened in assays for alteredproperties, including altered pH and temperature optimum, requirementfor divalent cations for enzymatic activity, mechanisms of enzymaticinhibition (e.g. salt, divalent cations, actin, heparin, proteases),substrate affinity and specificity (e.g. single-stranded DNA,double-stranded DNA, chromatin, NETs, plasmid DNA, mitochondrial DNA),localization upon secretion (e.g. membrane-bound, extracellular matrix),localization signals (e.g. nuclear localization signal, membraneanchor), glycosylation sites, disulfide-bonds and unpaired cysteines,compatibility with GMP-compliant in vitro expression systems (e.g.bacteria. yeast, mammalian cells), compatibility with carriers (e.g.PEGylation, Fc fragment, albumin), compatibility with GMP-compliantpurification methods (e.g. anion exchange resins, cation exchangeresins), toxicological profile, tissue penetration, pharmacokinetics andpharmacodynamics.

An extracellular DNASE with altered profile can provide a drug candidatefor diseases that are associated with NETs.

Example 2: Development of Building Block Engineering of DNase1-ProteinFamily Members

D1L3 features three sites that contain additional amino acids: theC-terminal tail starting after Q282 (NH2-SSRAFTBSKKSVTLRKKTKSKRS-COOH)(SEQ ID NO: 21), and at two sites within the enzyme at S79/R80 and atK226. The 23 amino acids of the C-terminal tail of D1L3 have beenattached to the C-terminus of D1. It was observed that the insertion ofan arginine-residue at position 226 of DNase1 (A226 T227insK) generateda D1-variant with reduced enzymatic activity to degrade dsDNA, while nosuch effect was observed with the substitution T227K. Thus, an insertionof a K/R-residue goes along with a risk of reducing D1 function. Theinsertion of a charged amino acid may influence the local proteinstructure. Given that D1 is a globular enzyme that comprises one aminoacid chain, it is conceivable that such local alteration may render thewhole enzyme inactive. Indeed, numerous non-conservative mutationsthroughout the D1 amino acid sequence inactivate the enzyme. Withoutbeing bound by theory, it was hypothesized that the transfer of localprotein structures by implanting not only single arginine and lysineresidues but also the neighboring amino acids sequences reduces the riskof inactivation. Conserved amino acids were searched within D1 and D1L3for in the vicinity of A226 T227insK that can be used as anchors for theinsertion. A D223/T224/T225 motif and a conserved T229 in D1 asN-terminal and C-terminal anchors, respectively were identified. 3 aminoacids within D1 (ATP) were replaced with 4 amino acids, including K226,from D1L3 (VKKS) in silico. Expression of the cDNA of the new D1-variant(A226_P228delinsVKKS) in HEK239 cells revealed a functionally activeenzyme with a similar dsDNA-degrading activity, when compared towild-type D1. The data suggest that the variable amino acids betweenconserved amino acids are interchangeable between D1 and D1L3.

We conceptualized a building block-technology to transfer enzymaticproperties from one member of the D1-protein family to another. Thefollowing cardinal steps characterize the technology (FIG. 4):

-   -   (1) Provide protein-protein alignment of donor and recipient        DNase    -   (2) Identify variable amino acid or amino acid sequence for        transfer (building block)    -   (3) Identify conserved amino acids in donor and recipient DNase        that are located up and downstream of building block (anchors),        respectively.    -   (4) Replace cDNA encoding for building block between C- and        N-anchors in recipient DNase, with cDNA between the anchors in        donor DNase.    -   (5) Synthesize cDNA of chimeric DNase, followed by in vitro/in        vivo expression into a recipient organism that is preferably        deficient in both donor and recipient DNase (e.g. CHO cells or        Dnase1^(−/−)Dnase113^(−/−) mice).

Example 3: Engineering DNase1 Variants Through Building Block Technology

A multiple-species alignment of D1 and D1L3 from human, mouse, rat, andchimpanzee (FIG. 5), showed that N- and C-terminal anchors are conservedamong these species. These anchor amino acids or amino acid sequencesflank 62 building blocks of variable amino acids and amino acidsequences, which include the amino acid sequence in D1 (ATP) and D1L3(VKKS) from building blocks #49 (FIGS. 6A-6B).

The transfer of these building blocks from D1L3 into D1 generatesD1-variants with the following mutations (FIGS. 6A-6B):1M_S22delinsMSRELAPLLLLLLSIHSALA, L23_A27delinsMRICS, I30_T32delinsVRS,E35_T36delinsES, M38_I47delinsQEDKNAMDVI, Q49_S52delinsKVIK, Y54C,I56_Q60delinsIILVM, V62_R63delinsIK, S65_K72delinsSNNRICPI,L74_N76delinsMEK, Q79_T84delinsRNSRRGIT, H86N, V88_V89delinsVI,E91_P92delinsSR, N96_S97delinsNT, R101Q, L103A, V105L,R107_Q110delinsKEKL, A113_S116delinsVKRS, Y118H, D120H,G122_N128delinsYQDGDA, T130S, N132S, A136_I137delinsFV, R139W,F141_F144delinsQSPH, E146_E149delinsAVKD, A151V, V153I,A157_A158delinsTT, G160_A162delinsETS, A164K, A168E, Y170_D171delinsVE,L174T, Q177_K179delinsKHR, G181_L182delinsKA, D184_L187delinsNFIF,R199_Q202delinsPKKA, S204_S205delinsKN, W209R, S211D, T213R, Q215V,P219G, S221_A222delinsQE, A226_P228delinsVKKS, H230N, V238_A239delinsLR,M241_A246delinsQEIVSS, D250K, A252_P254delinsNSV, N256D,A259_A260delinsKA, G262K, S264_L267delinsTEEE, Q269_I271delinsLDV,Y275F, V279_M280delinsFK, and K282delinsQSSRAFTNSKKSVTLRKKTKSKRS.

The following D1L3-variants are generated if the building blocks aretransferred from D1 to D1L3 (FIGS. 6A-6B): M1A20delinsMRGMKLLGALLALAALLQGAVS, M21 S25delinsLKIAA, V28_S30delinsIQT,E33_S34delinsET, Q36_I45delinsMSNATLVSYI, K47_K50delinsQILS, C52Y,I54_M58delinsIALVQ, I60_K61delinsVR, S63_I70delinsSHLTAVGK,M72_K74delinsLDN, R77_T84delinsQDAPDT, N86H, V88_I89delinsVV,S91_R92delinsEP, N96_T97delinsNS, Q101R, A103L, L105V,K107_L110delinsRPDQ, V113_S116delinsAVDS, H118Y, H120D,Y122_A127delinsGCEPCGN, V129T, S131N, 135F_136VdelinsAI, W138R,Q140_H143delinsFSRF, A145_D148delinsAVKD, V150A, I152A,T156_T157delinsAA, E159_S161delinsGDA, K163A, E167A, V169_E170delinsYD,T173L, K176_R178delinsQEK, K180_A181delinsGL, N183_F186delinsDVML,P198_A201delinsRPSQ, K203_N204delinsSS, R208W, D210S, R212T, V214Q,G218P, Q220_E221delinsSA, V225_S228delinsATP, N230H, L238_R239delinsVA,Q241_S246delinsMLLRGA, K250D, N252_V254delinsALP, D256N,K259_A260delinsAA, K262G, T264_E267delinsSDQL, L269_V271delinsQAI,F275Y, F279_K280delinsVM, and Q282_S305delinsK.

Next, we conceptualized a sequential approach to engineer D1-variantswith D1L3 activity that starts with the transfer of multiple adjacentbuilding blocks (clusters), continues with the transfer of individualbuilding blocks, and ends with a transfer of individual amino acids orthe combination of multiple building blocks into new chimeric enzymes(FIG. 7). This approach reduces the number of D1-D1L3-chimera in theinitial screening.

To test our method, we designed a total of 19 D1-variants comprisingeither individual building blocks or clusters of building block clusterfrom D1L3 (FIG. 6). These D1-variants feature the following amino acidmutations: 1M_S22delinsMSRELAPLLLLLLSIHSALA,L23_A27delinsMRICS/I30_T32delinsVRS/E35_T36delinsES,M38_I47delinsQEDKNAMDVI, Q49_S52delinsKVIK/Y54C/I56_Q60delinsIILVM,V62_R63delinsIK/S65_K72delinsSNNRICPI/L74_N76delinsMEK,Q79_T84delinsRNSRRGIT, H86NN88_V89delinsVI/E91_P92delinsSR,N96_S97delinsNT/R101Q/L103A/V105L,R107_Q110delinsKEKL/A113_S116delinsVKRS/Y118H/D120H,G122_N128delinsYQDGDA/T130S/N132S, A136_I137delinsFV,R139W/F141_F144delinsQSPH/E146_E149delinsAVKD/A151VN153I/A157_A158delinsTT/G160_A162delinsETS/A164K, A168E/Y170_D171delinsVE/L174T,Q177_K179delinsKHR/G181_L182delinsKA/D184_L187delinsNFIF,R199_Q202delinsPKKA/S204_S205delinsKN/W209R/S211D/T213R/Q215V/P219G/S221_A222delinsQE,A226_P228delinsVKKS,H230NN238_A239delinsLR/M241_A246delinsQEIVSS/D250K/A252_P254delinsNS V,N256D/A259_A260delinsKA/G262K/S264_L267delinsTEEE/Q269 I271delinsLDV,and Y275F/V279_M280delinsFK/K282delinsQSSRAFTNSKKSVTLRKKTKSKRS.

Next, we cloned the cDNA into an expression vector, which wastransfected into HEK293 cells. Analysis of the cell supernatants showeddsDNA degradation by all samples (FIG. 8). Furthermore, we observed thatthe transfer of building blocks (BB) 11, BB 12-14, BB 26, BB 41-48, andBB 49 from D1L3 to D1 resulted in enzymes with increased chromatindegrading activity. All these chimeric enzymes exhibited the same ormore activity to degrade dsDNA substrates than wild-type D1. Thebuilding blocks 11 and 49 from D1L3 contain R80/R81 and K227,respectively, which are not present in D1. The D1L3-BB cluster 41-48features 5 additional arginine and lysine residues than its counterpartin D1. These additional cationic amino acids may be responsible for thehyperactivity. The D1-building blocks 12-14 and 26 contain the aminoacid sequences H86 to R95 and A136 to V138 in SEQ ID NO: 1, whichincludes amino acid residues that are required for binding of theD1-inhibitor actin. Thus, replacement of these amino acid sequences withthe respective building blocks from D1L3, which do not interact withactin, likely generates actin-resistant variants of D1. We now combinedBB 11, 14, 26, 41-19 in one novel D1-variant. We observed that thecombination of these gain-of-function BBs increased the chromatindegrading of the D1 variant to levels of wild-type D1L3 (FIG. 8). Thus,the BB technology provides a robust method to generate hyperactive D1variants.

Example 4: Expression and Characterization of D1L3 with Basic DomainDeletion (BDD) in Chinese Hamster Ovarian (CHO) Cells and in Pichiapastoris

DNASE1 and DNASE1L3 preferentially cleave protein-free DNA andDNA-histone-complexes (i.e. chromatin), respectively. Previous studiessuggest that a basic domain (BD) at the C-terminus of DNASE1L3, which isabsent in DNASE1, is responsible for the distinct substratespecificities of both enzymes (Sisirak et al., Cell, 2016; Keyel,Developmental Biology, 2017).

To characterize the amino acids that are responsible forchromatin-degrading activity (“chromatinase” activity), wild-type D1L3was substituted with building block substitutions from D1, as disclosedin PCT/US2018/047084. The building block substitutions to D1L3 areselected from human D1 and result in variants of human D1L3, whichfeature the following mutations: M21_R22delinsLK, C24_S25delinsAA,V28_S30delinsIQT, S34T, Q36_V44delinsMSNATLVSY, K47_K50delinsQILS, C52Y,I55_M58delinsIALVQE, I60_K61delinsVR, N64_I70delinsHLTAVGK,M72_K74delinsLDN, R77_I83delinsQDAPD, N86H, I89V, S91_R92delinsEP, T97S,Q101R, A103L, L105V, K107_L110delinsRPDQ, V113_R115delinsAVD, H118Y,H120D, Y122_A127delinsGCEPCGN, V129T, S131N, F135_V136delinsAI, W138R,Q140_H143delinFSRF, A145_D148delinsEVRE, V150A, I152V,T156_T157delinsAA, E159_S161delinsGDA, K163A, E167A, V169_E170delinsYD,T173L, K176_R178delinsQEK, K180_A181 delinsGL, N183_F186delinsDVML,P198_A201delinsRPSQ, K203_N204delinsSS, R208W, D210S, R212T, V214Q,G218P, Q220_E221delinsSA, V225_S228delinsATP, N230H, L238_R239delinsVA,Q241_S246delinsMLLRGA, K250D, N252_V254delinsALP, D256N, K259A, K262G,T264_E267delinsSDQL, L269_V271delinsQAI, F275Y, F279_K280delinsVM,Q282_S205delinsK with respect to human D1L3, Isoform 1.

These 63 D1L3variants were screened for loss or gain ofchromatin-degrading activity. In brief, D1L3 variants were transientlyexpressed in CHO cells using an in vitro expression vector. Culturesupernatants were collected and tested for chromatin-degrading activityusing purified nuclei as a source of chromatin. As shown in FIG. 9, thebuilding block substitution #63 from D1 significantly improved thedegradation of high-molecular weight (HMW) chromatin to small fragments,when compared to wild-type D1L3. Building block substitution #63 causesthe mutation Q282 S305delinsK, which deletes the full C-terminal BD ofD1L3 from amino acid position 283 to 305 and replaces glutamine (Q) atposition 282 with lysine.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

Amino Acid Sequences of Wild-Type Human DNASES

DNASE1 (NP_005212.2): Signal Peptide, Mature Protein: SEQ ID NO: 1MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLKDNASE1-LIKE 1 (NP_006721.1): Signal Peptide; Mature Protein:SEQ ID NO: 2 MHYPTALLFLILANGAQAFRICAFNAQRLTLAKVAREQVMDTLVRILARCDIMVLQEVVDSSGSAIPLLLRELNRFDGSGPYSTLSSPQLGRSTYMETYVYFYRSHKTQVLSSYVYNDEDDVFAREPFVAQFSLPSNVLPSLVLVPLHTTPKAVEKELNALYDVFLEVSQHWQSKDVILLGDFNADCASLTKKRLDKLELRTEPGFHWVIADGEDTTVRASTHCTYDRVVLHGERCRSLLHTAAAFDFPTSFQLTEEEALNISDHYPVEVELKLSQAHSVQPLSLTVLLLLSLLSPQLCP AADNASE1-LIKE 2 (NP_001365.1): Signal Peptide, Mature Protein:SEQ ID NO: 3 MGGPRALLAALWALEAAGTAALRIGAFNIQSFGDSKVSDPACGSILAKILAGYDLALVQEVRDPDLSAVSALMEQINSVSEHEYSFVSSQPLGRDQYKEMYLFVYRKDAVSVVDTYLYPDPEDVFSREPFVVKFSAPGTGERAPPLPSRRALTPPPLPAAAQNLVLIPLHAAPHQAVAEIDALYDVYLDVIDKWGTDDMLFLGDFNADCSYVRAQDWAAIRLRSSEVFKWLIPDSADTTVGNSDCAYDRIVACGARLRRSLKPQSATVHDFQEEFGLDQTQALAISDHFPVEVTLKFHRDNASE1-LIKE 3; Isoform 1 (NP_004935.1): Signal Peptide, Mature Protein:SEQ ID NO: 4 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKT KSKRSDNASE1-LIKE 3, Isoform 2 (NP_001243489.1): SignalPeptide; Mature Protein: SEQ ID NO: 5MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNREKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRSDNASE2A (O00115): Signal Peptide; Mature Protein: SEQ ID NO: 6MIPLLLAALLCVPAGALTCYGDSGQPVDWFVVYKLPALRGSGEAAQRGLQYKYLDESSGGWRDGRALINSPEGAVGRSLQPLYRSNTSQLAFLLYNDQPPQPSKAQDSSMRGHTKGVLLLDHDGGFWLVHSVPNFPPPASSAAYSWPHSACTYGQTLLCVSFPFAQFSKMGKQLTYTYPWVYNYQLEGIFAQEFPDLENVVKGHHVSQEPWNSSITLTSQAGAVFQSFAKFSKFGDDLYSGWLAAALGTNLQVQFWHKTVGILPSNCSDIWQVLNVNQIAFPGPAGPSFNSTEDHSKWCVSPKGPWICVGDMNRNQGEEQRGGGILCAQLPALWKAFQPLVKNYQPCNGM ARKPSRAYKIDNASE2B (Q8WZ79): Signal Peptide; Mature Protein: SEQ ID NO: 7MKQKMMARLLRTSFALLFLGLFGVLGAATISCRNEEGKAVDWFTFYKLPKRQNKESGETGLEYLYLDSTTRSWRKSEQLMNDTKSVLGRTLQQLYEAYASKSNNTAYLIYNDGVPKPVNYSRKYGHTKGLLLWNRVQGFWLIHSIPQFPPIPEEGYDYPPTGRRNGQSGICITFKYNQYEAIDSQLLVCNPNVYSCSIPATFHQELIHMPQLCTRASSSEIPGRLLTTLQSAQGQKFLHFAKSDSFLDDIFAAWMAQRLKTHLLTETWQRKRQELPSNCSLPYHVYNIKAIKLSRHSYFSSYQDHAKWCISQKGTKNRWTCIGDLNRSPHQAFRSGGFICTQNWQIYQAF QGLVLYYESCK

Selected Amino Acid Sequences of Human Wild-Type DNASES

C-terminal tail of human DNASE1-LIKE 1 (NP_006721.1): SEQ ID NO: 8KLSQAHSVQPLSLTVLLLLSLLSPQLCPAAProline-rich extension of human DNASE1-LIKE 2 (NP_001365.1):SEQ ID NO: 9 SAPGTGERAPPLPSRRALTPPPLPAAAQNLVLIPLC-terminal tail of human DNASE1-LIKE 3; Isoform 1 (NP_004935.1):SEQ ID NO: 10 SSRAFTNSKKSVTLRKKTKSKRSInternal sequence of human DNASE1-LIKE 3; Absentin Isoform 2 (NP_004935.1): SEQ ID NO: 11RNSRRGITYNYVISSRLGRNTYKEQYAFLYK

Carrier Protein

Human Albumin (P02768): Signal Peptide + Propeptide; Mature Protein:SEQ ID NO: 12 MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGL

Amino Acid Sequences of Human ALBUMIN-DNASE-Fusion Proteins

Albumin-Linker-DNASE1;Signal Peptide + Propeptide, Albumin, Flexible Linker, mature DNASE1:SEQ ID NO: 13 MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQATSDHYPVEVMLK Albumin-Linker-DNASE1-LIKE 1;Signal Peptide + Propeptide, Albumin, Flexible Linker, matureDNASE1-LIKE 1: SEQ ID NO: 14MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSFRICAFNAQRLTLAKVAREQVMDTLVRILARCDIMVLQEVVDSSGSAIPLLLRELNRFDGSGPYSTLSSPQLGRSTYMETYVYFYRSHKTQVLSSYVYNDEDDVFAREPFVAQFSLPSNVLPSLVLVPLHTTPKAVEKELNALYDVFLEVSQHWQSKDVILLGDFNADCASLTKKRLDKLELRTEPGFHWVIADGEDTTVRASTHCTYDRVVLHGERCRSLLHTAAAFDFPTSFQLTEEEALNISDHYPVEVELKLSQAHSVQPLSLTVLLLLSLLSPQLCPAA Albumin-Linker-DNASE1 -LIKE 2;Signal Peptide + Propeptide, Albumin, Flexible Linker, matureDNASE1-LIKE 2: SEQ ID NO: 15MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSLRIGAFNIQSFGDSKVSDPACGSIIAKILAGYDLALVQEVRDPDLSAVSALMEQINSVSEHEYSFVSSQPLGRDQYKEMYLFVYRKDAVSVVDTYLYPDPEDVFSREPFVVKFSAPGTGERAPPLPSRRALTPPPLPAAAQNLVLIPLHAAPHQAVAEIDALYDVYLDVIDKWGTDDMLFLGDFNADCSYVRAQDWAAIRLRSSEVFKWLIPDSADTTVGNSDCAYDRIVACGARLRRSLKPQSATVHDFQEEFGLDQTQALAISDHFPVEVTLKFHR Albumin-Linker-DNASE1-LIKE 3;Signal Peptide + Propeptide, Albumin, Flexible Linker, matureDNASE1-LIKE 3: SEQ ID NO: 16MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRSAlbumin-Linker-DNASE1-LIKE 3, Isoform 2;Signal Peptide + Propeptide, Albumin, Flexible Linker, matureDNASE1-LIKE 3 Isoform 2: SEQ ID NO: 17MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNREKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTL RKKTKSKRSAlbumin-Linker-DNASE2A;Signal Peptide + Propeptide, Albumin, Flexible Linker, mature DNASE2A:SEQ ID NO: 18 MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSCYGDSGQPVDWFVVYKLPALRGSGEAAQRGLQYKYLDESSGGWRDGRALINSPEGAVGRSLQPLYRSNTSQLAFLLYNDQPPQPSKAQDSSMRGHTKGVLLLDHDGGFWLVHSVPNFPPPASSAAYSWPHSACTYGQTLLCVSFPFAQFSKMGKQLTYTYPWVYNYQLEGIFAQEFPDLENVVKGHHVSQEPWNSSITLTSQAGAVFQSFAKFSKFGDDLYSGWLAAALGTNLQVQFWHKTVGILPSNCSDIWQVLNVNQTAFPGPAGPSFNSTEDHSKWCVSPKGPWTCVGDMNRNQGEEQRGGGTLCAQLPALWKAFQPLVKNYQPCNGMARKPSRAYKI Albumin-Linker-DNASE2B;Signal Peptide + Propeptide, Albumin, Flexible Linker, mature DNASE2B:SEQ ID NO: 19 MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSATISCRNEEGKAVDWFTFYKLPKRQNKESGETGLEYLYLDSTTRSWRKSEQLMNDTKSVLGRTLQQLYEAYASKSNNTAYLIYNDGVPKPVNYSRKYGHTKGLLLWNRVQGFWLIHSIPQFPPIPEEGYDYPPTGRRNGQSGICITFKYNQYEAIDSQLLVCNPNVYSCSIPATFHQELIHMPQLCTRASSSEIPGRLLTTLQSAQGQKFLHFAKSDSFLDDIFAAWMAQRLKTHLLTETWQRKRQELPSNCSLPYHVYNIKAIKLSRHSYFSSYQDHAKWCISQKGTKNRWTCIGDLNRSPHQAFRSGGFICTQNWQIYQAFQGLVLYYESCK SEQ ID NO: 20 (Intentionally left blank)SEQ ID NO: 21 (Intentionally left blank) SEQ ID NO: 23(Intentionally left blank) SEQ ID NO: 24 (Intentionally left blank)SEQ ID NO: 25 (Intentionally left blank) SEQ ID NO: 26(Intentionally left blank) SEQ ID NO: 27 (Intentionally left blank)SEQ ID NO: 28 (Intentionally left blank)

Human DNASE1L3 Variants

SEQ ID NO: 29 (Intentionally left blank)DNASE1L3, Q282_S305delinksK (Signal Peptide; Mature Protein):SEQ ID NO: 30 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNRLRTDPRFVWLIGDQEDTTVKKSINCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLKMurine DNase1L3 (O55070): Amino acid sequence (SignalPeptide; Mature Protein): SEQ ID NO: 31MSLHPASPRLASLLLFILALHDTLALRLCSFNVRSFGASKKENHEAMDIIVKIIKRCDLILLMEIKDSSNNICPMLMEKLNGNSRRSTTYNYVISSRLGRNTYKEQYAFVYKEKLVSVKTKYHYHDYQDGDTDVFSREPFVVWFHSPFTAVKDFVIVPLHTTPETSVKEIDELVDVYTDVRSQWKTENFIFMGDFNAGCSYVPKKAWQNIRLRTDPKFVWLIGDQEDTTVKKSTSCAYDRIVLCGQEIVNSVVPRSSGVFDFQKAYDLSEEEALDVSDHFPVEFKLQSSRAFTNNRKSVSLKKRKKGNRSRat DNase1L3 (O89107): Amino acid sequence (Signal Peptide;Mature Protein): SEQ ID NO: 32MSLYPASPYLASLLLFILALHGALSLRLCSFNVRSFGESKKENHNAMDIIVKIIKRCDLILLMEIKDSNNNICPMLMEKLNGNSRRSTTYNYVISSRLGRNTYKEQYAFLYKEKLVSVKAKYLYHDYQDGDTDVFSREPFVVWFQAPFTAAKDFVIVPLHTTPETSVKEIDELADVYTDVRRRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPNFVWLIGDQEDTTVKKSTSCAYDRIVLRGQEIVNSVVPRSSGVFDFQKAYELSEEEALDVSDHFPVEFKLQSSRAFTNSRKSVSLKKKKKGSRSChimpanzee DNase1L3 (H2QMU7): Amino acid sequence (SignalPeptide; Mature Protein): SEQ ID NO: 33MSRELTPLLLLLLSIHSTLALRICSFNVRSFGESKQEDQNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSINCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS Olive baboon DNase1L3 (A0A2I3NFJ3): Amino acid sequence(Signal Peptide; Mature Protein): SEQ ID NO: 34MSQELAPLLLLLLSIHSALALRICSFNVRSFGESKQEDQNAMDVIVKVIKRCDIMLLMEIKDSNNRICPVLMEKLNGNSRRGIMYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDVDVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVDVYMDMKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKRSTKCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTVRKKTKSKRSRabbit DNase1L3 (A0A2I3NFJ3): Amino acid sequence (SignalPeptide; Mature Protein): SEQ ID NO: 35MSLGMSPASLLLLLLCLHGALALKLCSFNVRSFGYSKRENRQAMDVIVKIIKRCDIILLMEIKDSNNMICPTLMEKLNGNSRRGITYNYVISSRLGRNVYKEQYAFLYKEKLVTVKKNYLYHDYEAGDADAFSREPYVVWFQSPFTAVKDFVIVPLHTSPEASVKEIDELVDVYMDVKRRWNAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDEEDTTVKKSTSCAYDRIVLRGQDIIRSVVPDSNGVFDFRKAYKLTEEEALDVSDHFPVEFKLQSSTAFTNSKKSVQPRKKAKAKRSDog DNase1L3 (F1P9C1): Amino acid sequence (Signal Peptide;Mature Protein): SEQ ID NO: 36MPRLPAFLLFLLLSISSALALRLCSFNVRSFGGAKRENKNAMDVIVKVIKRCDIILLMEVKDSNNMICPTLLEKLNGNSRRGIKYNYVISSRLGRNTYKEQYAFLYKEKLVSVKKYYLYHDYQAGDADVFSREPFVVWFQSPFTAVKDFVIVPLHTTPEASVKEIDELVDVYLDVKRRWKAENFIFMGDFNAGCSYVPKKAWKIIRLRTDPGFVWLIGDQEDTTVKSSTHCAYDRIVLRGPEIIRSVVPRSNSTFDFQKAFLLTEEEALNVSDHFPVEFKLQSSRAFTNSKKSISPKKKKVRHP Pig DNase1L3 (A0A287B132): Amino acid sequence (predictedSignal Peptide; Mature Protein): SEQ ID NO: 37MSQLLVSLMLLLLSTHSSLALRICSFNVRSFGESKKANCNAMDVIVKVIKRCDIILLMEIKDSNNMICPTLMEKLNGNSRRSVTYNYVISSRLGRNTYKEQYAFLYKEKLVSVKKSYLYHDYQSGDADVFSREPFVVWFQSPYTAVKDFVIIPLHTTPETSVKEIDELVDVYLDVKRRWEAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPMFIWLIKDQEDTTVKKSINCAYDRIVLRGQEIVSSVVPGSNSIFDFQKAYRLTEEKVRLSFCLSVSPSGEDGVVSPRGIQATTGDTLGHLTLSFKANDSLTGuinea pig DNase1L3 (A0A286XK50): Amino acid sequence(Signal Peptide; Mature Protein): SEQ ID NO: 38MSQTRPSLLLLLLAIHGALALKLCSFNVRSFGESKKQNQNAMDVIVKIIKRCDLMLLMEIKDSHNRICPMLMEKLNGNSRRGTTYNYVISSRLGRNTYKEQYAFLYKEKLVTVKDNYLFHDEDADVFSREPYVVWFQSPHTAVKDFVIVPLHTTPETSVKEIDELADVYTDVQRQWKVANFIFMGDFNAGCSYVPKKAWKNIRLRTDPKFVWLIADDEDTTVKKSTSCAYDRIVLRGQEIVNSVVPNSNGVFDFQKAYQLSEEQALEVSDHFPVEFKLQSERAFTNNKKS VSLKKKKKANRSCow DNase1L3 (F1MGQ1): Amino acid sequence (Signal Peptide;Mature Protein): SEQ ID NO: 39MPLPLACLLLLLLSTHSALALKICSFNVRSFGESKKANCNAMDVIVKVIKRCDIILLMEIKDSSNRICPTLMEKLNGNSRKGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKQSYLYHDYQAGDADVFSREPFVVWFQSPYTAVKDFVIVPLHTTPETSVREIDELADVYTDVKRRWNAENFIFMGDFNAGCSYVPKKAWKDIRLRTDPKFVWLIGDQEDTTVKKSINCAYDRIVLRGQNIVNSVVPQSNLVFDFQKAYRLSESKALDVSDHFPVEFKLQSSRAFTNSKKSVSSKKKKKTSHA Elephant DNase1L3 (G3SXX1) : Amino acid sequence (SignalPeptide; Mature Protein): SEQ ID NO: 40RSARMSQSLPALLLLLLLSVHGTLALRVCSFNVRSFGETKRENQKVMDIIVKIIKRCDIMLLMEIKDSNNRICPMLLKRLNGNSRRGIKYNYVISPRLGRNAYKEQYAFLYMEKLLSVKKSYVYGDNQNGDADVFSREPFVTWFQSPHTAVKDFVIVPLHTTPETSIKEIDELVDVYMDVKKRWNAQNFIFMGDFNAGCSYVPKKSWRNIRLRTDPGFVWLIGDQEDTTVKESTNCAYDRIVLRGQIISSVVPNSNSIFNFQKAYELSEEEALNISDHFPVEFKLQSSRAIINSKKSVSPKKKKKAKSS

Linker Sequences

SEQ ID NO: 41 GGGGS SEQ ID NO: 42 GGGGSGGGGSGGGGS SEQ ID NO: 43APAPAPAPAPAPAP SEQ ID NO: 44 AEAAAKEAAAKA SEQ ID NO: 45 SGGSGSSSEQ ID NO: 46 SGGSGGSGGSGGSGSS SEQ ID NO: 47SGGSGGSGGSGGSGGSGGSGGSGGSGGSGS SEQ ID NO: 48GGSGGSGGSGGSGGSGGSGGSGGSGGSGS

Activatable Linker Sequences

FXIIa-susceptible linker (Factor XI peptide): SEQ ID NO: 49CTTKIKPRIVGGTASVRGEWPWQVTFXIIa-susceptible linker (Prekallikrein peptide): SEQ ID NO: 50 STRIVGGFXIIa-susceptible linker (Prekallikrein peptide): SEQ ID NO: 51VCTTKTSTRIVGGTNSSWGEWPWQVS

What is claimed is:
 1. A method for making a DNASE therapeuticcomposition for treating a disorder associated with pathological levelsof extracellular chromatin or NETs, the method comprising: evaluating aplurality of extracellular DNASE variants having building blocksubstitutions and/or half-life extension moiety for one or morecharacteristics selected enzymatic activity, nucleic acid substratepreference, potential for recombinant expression in prokaryotic oreukaryotic host cells, immunogenic potential in humans and animals, andpharmacodynamics in animal models; selecting a DNASE variant having adesired enzymatic, physical, immunological and/or pharmacodynamicsprofile, and formulating the selected DNASE variant for administrationto a patient.
 2. The method of claim 1, wherein at least 5, or at least10, or at least 20, or at least 50 extracellular DNASE variants areevaluated.
 3. The method of claim 1 or 2, wherein the variants areselected from one or more of D1 variants, D1L1 variants, D1L2 variants,D1L3 isoform 1 variant, D1L3 isoform 2 variants, D2A variants, and D2Bvariants.
 4. The method of claim 3, wherein the method evaluates one ormore D1L1 variants having a building block substitution, fusion orconjugation to a half-life extension moiety, and/or substitution from anon-human D1L1.
 5. The method of claim 3, wherein the method evaluatesone or more D1L2 variants having a building block substitution, fusionor conjugation to a half-life extension moiety and/or substitution froma non-human D1L2.
 6. The method of claim 3, wherein the method evaluatesone or more D1L3 variants having a building block substitution, fusionor conjugation to a half-life extension moiety, and/or substitution froma non-human D1L3.
 7. The method of claim 3, wherein the method evaluatesone or more D1L3-2 variants having a building block substitution, fusionor conjugation to a half-life extension moiety, and/or substitution froma non-human D1L3-2.
 8. The method of claim 3, wherein the methodevaluates one or more D2A variants having a building block substitution,fusion or conjugation to a half-life extension moiety, and/orsubstitution from a non-human D2A.
 9. The method of claim 3, wherein themethod evaluates one or more D2B variants having a building blocksubstitution, fusion or conjugation to a half-life extension moiety,and/or substitution from a non-human D2B.
 10. The method of claim 3,wherein the method evaluates one or more D1 variants having a buildingblock substitution, fusion or conjugation to a half-life extensionmoiety and/or substitution from a non-human D1.
 11. The method of claim10, where the D1 variants comprises an amino acid sequence that is atleast 80% identical to the enzyme defined by SEQ ID NO:
 1. 12. Themethod of claim 11, wherein the D1 variants include variants having oneor more mutations selected from non-human D1 enzymes, and which areselected from: K24R, I25M, Q31R, T32S, E35D, V44S, V44T, V44A, S45V,S45K, S45N, S45H, Q49K, Q49R, S52Q, S52R, R53L, I56V, A57V, L58V, V59I,560T, T68V, D75N, N76E, N76K, N76T, N76E, N76Y, N76S, Q79R, Q79E, D80K,D80H, A81K, A81I, A81D, P82A, P82T, D83N, D83G, T84N, T84A, Y85F, H86R,Y87F, Y87H, V88I, V89I, V89A, N96K, N96R, N96S, S97T, R101Q, V105L,Y106F, D109S, Q110R, Q110K, A113V, A113I, S114L, S116T, Y108Q, Y108H,Y108L, P125S, N128T, T130S, N132S, N132A, A136S, I137V, R139K, F141S,F141H, S142C, R143P, R143H, F144Y, F144S, F144L, V147K, V147Q, R148Q,R148S, E149K, I152V, P154A, A157S, G160E, G160T, G160L, G160S, D161E,V163A, S164S, D167N, A168S, D175N, Q177W, Q177R, E178Q, E178K, E178H,G181D, G181H, E183Q, E183N, V185I, M186V, L187F, G194D, C195Y, R199T,R199A, R199S, P200S, P200A, P200T, P200L, Q202H, S204A, W209R, T210M,T210E, P212S, T213A, T213I, T213P, Q215K, Q215R, P219L, S221T, S221N,A226V, A226S, T227S, T227K, P228S, H230N, A232P, M241T, M241A, M241P,M241S, R244Q, G245D, G245A, G245H, G245R, G245S, D250N, D250S, D250E,D250G, L253V, L253A, L253M, N256D, A259V, A260E, Y261F, G262R, S264T,D265N, D265S, D265E, Q266E, L267M, L267T, Q269E, Q269L, M280T, M280A,K282R, K282A, K282T, K282insK, and K282insR.
 13. The method of claim 11,wherein at least one D1 variant has a building block substitutionselected from human D1L1.
 14. The method of claim 11, wherein at leastone D1 variant has a building block substitution selected from humanD1L2.
 15. The method of claim 11, wherein at least one D1 variant has abuilding block substitution selected from human D1L3.
 16. The method ofclaim 11, wherein at least one D1 variant has a building blocksubstitution selected from human D1L3-2.
 17. The method of claim 4,wheren the D1L1 variants comprises an amino acid sequence that is atleast 80% identical to the enzyme defined by SEQ ID NO:
 2. 18. Themethod of claim 17, wherein the D1L1 variants include variants havingone or more mutations selected from non-human D1L1 enzymes, and whichare selected from: A26T, Q27H, A32T, A32S, V34L, A35T, A35I, R36K, Q38S,Q38E, Q38H, Q38Y, Q38P, Q38D, M40K, M40L, T42I, L43F, R45Q, R45K, L47V,M53T, S61A, S62T, G63Q, G63D, G63N, G63S, S64N, S64A, S64K, S64T, A65T,P66L, P66S, L68F, R71Q, R71E, E72K, N74S, R75K, F76Y, D77K, D77Q, D77Y,D77G, G78A, G78S, G78N, G78D, G80R, G80K, P81S, P81F, P81C, S83R, T84F,T84S, L85H, S86N, S86K, P88S, P88D, Q89L, Q89M, S93N, S93G, T94A, M96V,M96K, T98K, V100A, F102I, H106D, K107R, K107E, K107R, T108A, Q109E,V110L, L111R, S112N, S112D, S112E, S113F, V115Q, V115L, V115M, N117D,N117E, N117P, N177S, E119T, E119Q, E119K, V122I, V122L, A124T, A130G,A130C, Q131H, Q131W, S133T, L134F, P135R, N137D, N137K, V138T, V138I,L142V, V143A, A153D, K156P, K156N, K156T, L159K, Y162H, D163E, D163T,E167D, V168A, S169Y, S169A, Q170R, Q170G, H171R, S174N, S174T, K175E,K175Q, S176N, V177M, V177I, A188T, T191A, T191N, D196K, D196N, D196S,D196A, D196G, E199L, E199A, E199V, E203K, E203D, E203Q, P204A, P204V,P204T, H207R, H207S, V209A, I210V, A211P, E214D, E241V, H223N, T225A,V229I, L231V, L231M, E234Q, E234V, R235G, R235T, R235L, C236L, R237Q,S238M, S238K, S238G, L240M, H241K, H241Q, H241S, H241R, T242A, T242S,T242N, T242G, A244T, D247N, T240K, T250R, Q250Q, S251T, S251R, Q252R,Q252G, T255N, T255S, E258Q, E259Q, N261R, N261K, M261T, I262V, E271D,K273S, K273N, K273D, K273A, L274del, S275Q, S275K, S275R, Q276A, A277T,A277V, H278P, H278Q, S279G, S279N, S279R, C279S, I280V, A280V, Q281P,Q281L, L283H, L283P, S284Y, S284C, S284H, S284G, T286A, T286S, T286V,V287T, V287A, F287F, G287V, L288A, L288S, L289S, L289V, L289M, S292L,S292P, S295P, S295T, S295A, P296S, Q297E, L298C, C299D, C299G, C299S,P300L, A301Q, A301V, and A302M.
 19. The method of claim 17, wherein atleast one D1L1 variant has a building block substitution selected fromhuman D1.
 20. The method of claim 17, wherein at least one D1L1 varianthas a building block substitution selected from human D1L2.
 21. Themethod of claim 17, wherein at least one D1L1 variant has a buildingblock substitution selected from human D1L3.
 22. The method of claim 17,wherein at least one D1L1 variant has a building block substitutionselected from human D1L3-2.
 23. The method of claim 17, wherein the D1L1variant contains one or more amino acid substitutions, additions, ordeletions in the C-terminal tail.
 24. The method of claim 5, wheren theD1L2 variants comprises an amino acid sequence that is at least 80%identical to the enzyme defined by SEQ ID NO:
 3. 25. The method of claim24, wherein the D1L2 variants include variants having one or moremutations selected from non-human D1L2 enzymes, and which are selectedfrom: L22K, I24V, I29V, S35N, S35H, S35R, S35T, V37A, S38L, A41D, A41V,A41G, G431, S44G, S44i, I45V, K48Q, L55I, L55V, A56T, A56M, P64A, 570D,570T, A71T, A71L, A71S, A71V, M73L, E74Q, N77H, S78R, E81K, E81R, E83N,S85G, S85N, Q90E, Q90K, Q96H, F103Y, V104I, K107D, A109V, A109T, A109K,V110A, V113L, V113M, D114S, D114E, L117Q, P119S, E122G, V124A, V124F,S126N, E128D, F134V, A136V, A136T, G138S, G138R, T139S, T139C, S148C,A151P, P154A, A159P, A160G, A161P, A161T, Q162D, Q162K, Q162R, Q162T,N163K, N163E, L164V, L164F, I167V, H174N, Q175H, A178T, A178V, D192N,G195N, T196S, D198V, M199L, M199I, S210K, R213K, Q215H, A218P, A219S,E226Q, V227I, S243T, A252V, C253S, A255S, A255V, R256H, L257M, R259K,S260T, L261V, Q264H, T267S, T267A, D270N, G276D, G276S, T280S, T280D,T280A, A284C, I286V, L295F, F297S, F297T, F297P, H298R, and R299del. 26.The method of claim 24, wherein at least one D1L2 variant has a buildingblock substitution selected from human D1.
 27. The method of claim 24,wherein at least one D1L2 variant has a building block substitutionselected from human D1L1.
 28. The method of claim 24, wherein at leastone D1L2 variant has a building block substitution selected from humanD1L3.
 29. The method of claim 24, wherein at least one D1L2 variant hasa building block substitution selected from human D1L3-2.
 30. The methodof claim 24, wherein at least one D1L2 protein variant contains one ormore amino acid substitutions, additions, or deletions in theproline-rich extension domain.
 31. The method of claim 6, wheren theD1L3 variants comprises an amino acid sequence that is at least 80%identical to the enzyme defined by SEQ ID NO:
 4. 32. The method of claim31, wherein the D1L3 variants include variants having one or moremutations selected from non-human D1L3 enzymes, and which are selectedfrom: M21L, K22R, I22L, I22V, E33A, E33Y, E33G, S34A, S34T, Q36K, Q36R,E37A, E37Q, D48N, K39Q, K39H, K39R, K39C, N40E, N40Q, N40K, A41V, V44I,V53I, I53L, I54M, V57L, I60V, N64S, H64S, R66N, R66M, I70V, I70M, I70T,M72L, E73K, K74R, R77G, R81K, G82S, I83V, I83T, T84M, T84K, S91P, T91V,T91A, L105V, K107M, V111L, S112T, R115T, R115A, R115K, R115D, R115Q,S116K, S116N, S116Y, H118L, H118V, Y119F, H120G, Y122N, Q123E, D124A,D124S, D124N, G125E, A127V, A127T, V129A, F135Y, V137T, Q140H, S141A,H143F, H143Y, V146A, I152V, T157S, T160A, V162I, K163R, V169A, E170D,T173M, T173L, V175M, K176R, K176Q, H177S, H177R, R178Q, K180E, K180N,K181T, K181V, E183A, E183Q, A201S, K203Q, K203R, R212K, R212N, R212G,R212M, V214I, G218K, G218A, Q220E, Q220D, K227R, K227S, K227E, N239K,N239S, N239H, R239C, Q241P, E242D, E242N, V244I, S245N, S245R, K250R,K250D, K250R, K250G, K250N, K250Q, N252S, S253G, S253L, V254T, V254I,D256N, Q258R, Y261F, K262D, K262E, K262L, K262R, K262Q, T264S, E266S,E267K, E267Q, E267K, D270N, D270E, V271I, S282E, R285T, F287I, S290N,K291R, V294I, T295S, T295Q, L296V, L296P, L296S, R297K, K299R, T300K,T300A, S302G, S302A, S302V, S302T, K303N, K303S, K303R, R304H, R304S,S305P, S305T, and S305A.
 33. The method of claim 31, wherein at leastone D1L3 variant has a building block substitution selected from humanD1.
 34. The method of claim 31, wherein at least one D1L3 variant has abuilding block substitution selected from human D1L1.
 35. The method ofclaim 31, wherein at least one D1L3 variant has a building blocksubstitution selected from human D1L2.
 36. The method of claim 31,wherein at least one D1L3 variant contains one or more amino acidsubstitutions, additions, or deletions in the C-terminal tail.
 37. Themethod of claim 31, wherein at least one D1L3 variant contains one ormore amino acid substitutions, additions, or deletions in the internalsequence defined by SEQ ID NO: 11, and which is optionally deleted inwhole or in part.
 38. The method of claim 7, wheren the D1L3-2 variantscomprise an amino acid sequence that is at least 80% identical to theenzyme defined by SEQ ID NO:
 5. 39. The method of claim 38, wherein theD1L3-2 variants include variants having one or more mutations selectedfrom non-human D1L3 enzymes, and which are selected from: M21L, K22R,I22L, I22V, E33A, E33Y, E33G, S34A, S34T, Q36K, Q36R, E37A, E37Q, D48N,K39Q, K39H, K39R, K39C, N40E, N40Q, N40K, A41V, V44I, V53I, I53L, I54M,V57L, I60V, N64S, H64S, R66N, R66M, I70V, I70M, I70T, M72L, E73K, K74R,R77G, V81L, S82T, R85T, R85A, R85K, R85D, R85Q, S86K, S86N, S86Y, H88L,H88V, Y89F, H90G, Y92N, Q93E, D94A, D94S, D94N, G95E, A97V, A97T, V99A,F105Y, V107T, Q110H, S111A, H113F, H113Y, V116A, I122V, T127S, T130A,V132I, K133R, V139A, E140D, T143M, T143L, V145M, K146R, K146Q, H147S,H147R, R148Q, K150E, K150N, K151T, K151V, E153A, E153Q, A171S, K173Q,K173R, R182K, R182N, R182G, R182M, V184I, G188K, G188A, Q190E, Q190D,K197R, K197S, K197E, N209K, N209S, N209H, R209C, Q211P, E212D, E212N,V214I, S215N, S215R, K220R, K220D, K220R, K220G, K220N, K220Q, N222S,S223G, S223L, V224T, V224I, D226N, Q228R, Y231F, K232D, K232E, K232L,K232R, K232Q, T234S, E236S, E237K, E237Q, E237K, D240N, D240E, V241I,S252E, R255T, F257I, S260N, K261R, V264I, T265S, T265Q, L266V, L266P,L266S, R267K, K269R, T270K, T270A, S272G, S272A, S272V, S272T, K273N,K273S, K273R, R274H, R274S, S275P, S275T, and S275A.
 40. The method ofclaim 38, wherein at least one D1L3-2 variant has a building blocksubstitution selected from human D1.
 41. The method of claim 38, whereinat least one D1L3-2 variant has a building block substitution selectedfrom human D1L1.
 42. The method of claim 38, wherein at least one D1L3-2variant has a building block substitution selected from human D1L2. 43.The method of claim 38, wherein at least one D1L3-2 variant contains oneor more amino acid substitutions, additions, or deletions in theC-terminal tail domain defined by SEQ ID NO:
 11. 44. The method of claim8, wheren the D2A variants comprise an amino acid sequence that is atleast 80% identical to the enzyme defined by SEQ ID NO:
 6. 45. Themethod of claim 44, wherein the D2A variants include variants having oneor more mutations selected from non-human D2A enzymes, and which areselected from: Q25R, L38H, L38N, R39S, R39T, G40S, G42R, E43D, A44T,A44K, A44V, A45P, A45I, R47K, R47N, R47S, Q50T, Q50M, Q50R, L54M, L54F,E56Q, S57N, S57H, S57E, G59D, G59E, G60D, R62Q, R62S, R65V, R65A, A66G,L67Y, L67H, L67F, L67S, N69D, P71S, P71K, P71T, E72D, E72T, V75L, R77L,Q80L, R84Q, S85K, S85N, T87S, T87N, L93V, Q101K, P102S, P102Y, S103R,K104S, K104E, K104G, A105S, Q106R, Q106K, D107H, S109T, M110G, M110S,M110N, R111H, H122Q, D123E, V129I, N134R, P137S, P138R, A139S, A142G,A143V, S145T, H148P, S149N, S149G, C151Q, C151R, I152K, Y153F, L158I,F162L, F164L, A165I, A165S, S168A, S168P, S168L, K169R, K169G, K169D,K169N, M170I, G171S, K172R, W180L, W180M, N183D, Y184H, Q185K, Q185R,I180F, I180D, Q192R, E193K, F194L, D196Y, N199I, N199E, V201I, V201T,G203N, G203Q, S207L, S207R, Q208H, Q208R, E209G, I215V, T216I, Q220R,Q220K, A221K, A223T, V224T, V224S, F231C, S232G, K233N, A244S, A245E,T249S, N250I, H257Q, H257P, T259S, V260P, V260S, V260A, D269G, I270A,I270I, I270V, W271Y, W271H, W271Q, Q272K, Q272H, V273I, L274F, N275D,N277T, Q278E, I279I, A280G, A285S, G286R, P287L, S288T, S288A, S288N,N290S, S291A, S301A, S301T, K303Q, K303E, G304R, T307A, T307V, Q316K,G317A, G317R, E319I, Q320H, L326V, A328T, L330V, L330M, A332S, L333F,Q338R, Q338K, P339S, N343D, N343A, Y344W, Y344C, Q345K, and Q345E. 46.The method of claim 44, wherein at least one D2A variant has a buildingblock substitution selected from human D2B.
 47. The method of claim 9,wheren the D2B variants comprise an amino acid sequence that is at least80% identical to the enzyme defined by SEQ ID NO:
 7. 48. The method ofclaim 47, wherein the D2B variants include variants having one or moremutations selected from non-human D2B enzymes, and which are selectedfrom: A28P, A28T, T29E, T29V, T29K, S31A, R33I, N34S, E36Y, E36D, A37P,T44I, T44A, T44V, K50R, R51Q, R51K, Q52T, N53S, N53D, N53E, K54R, E55A,E55G, S56G, G57E, G57T, G57R, T59A, T59M, E62Q, E62D, E62G, T70R, T70M,T701, R71Q, S72T, R74N, R74S, R74K, K75R, E77L, E77H, E77K, Q78Y, Q78H,Q78L, M80I, M80V, D82T, D82S, D82A, T83S, K84R, K84D, V86A, V86S, Q92E,Q93H, E96D, A97T, Y98H, Y98N, Y98C, A99D, A99H, S100A, S100F, K101E,S102T, S102N, S102D, N104D, N104S, L108V, I109L, G113A, V114I, K116G,K116A, P117S, V118A, N119T, N119G, N119S, Y120C, R122G, K123Q, K123N,Y124F, T127A, L132V, V136T, V136I, I145V, Q147K, Q147R, I151V, I151T,E154H, E154K, D157E, P160T, P160S, T161S, R164Q, N165Y, N165H, G166A,S168T, S168A, S168N, I170L, I170M, F174L, K175G, K175R, N178S, Y179F,A181E, A181T, S184F, V188I, C189L, C189F, C189Y, N190Q, V192I, S195R,S197F, A200S, A200N, A200T, T201I, T201A, H203R, Q204W, Q204M, E205K,I207V, I207F, H208Y, H208Y, M209L, Q211R, L212M, T214A, R215K, R215G,A216S, S217T, S217H, S218A, S219L, E220K, G223V, G223S, R224Q, L225Y,L225R, L225H, T227A, T228E, T228V, T228S, Q230H, Q233R, Q235L, K236N,K236S, L238V, L238I, S243F, D244S, D244T, S245F, F246Y, L247T, L247H,A252T, A252V, A253G, M255I, R258K, R258H, R258Q, T261V, T265A, T265V,E266Q, T267S, R270K, R272K, R272N, R272G, Q273H, Y283H, C285I, I288V,A290S, K292G, K292R, L293V, L293G, L293I, R295G, R295S, R295L, R295H,H296K, H296Q, Y298D, S300P, Y302R, Y302H, Q303H, A306S, I310V, Q312I,Q312T, Q312R, Q312L, G314D, G314R, T315S, K316A, K316Q, N317A, R318H,P329L, H330Y, F333L, F333S, S335G, T341S, T341N, Q342K, W344H, W344R,W344Q, Q345H, Q345Y, Q345R, Q345N, Q349H, Q351H, Q351D, Q351E, G352K,G352R, V354Y, L355S, Y356R, Y356H, Y357H, E358G, E358A, S359F, S359N,S359D, and K361N.
 49. The method of claim 47, wherein at least one D2Bvariant has a building block substitution selected from human D2A. 50.The method of any one of claims 1 to 49, wherein the variants comprisean N-terminal or C-terminal fusion to a half-life extending moiety. 51.The method of claim 50, wheren the half-life extending moieties areindependently selected from albumin, transferrin, Fc, and elastin-likeprotein.
 52. The method of claim 50, wherein the variants comprise anN-terminal fusion of human albumin with a linking sequence.
 53. Themethod of any one of claims 1 to 49, wherein the variants comprisevariants with one or more polyethylene glycol (PEG) moieties.
 54. Themethod of any one of claims 1 to 53, wherein the DNASE variants areevaluated in assays for: altered properties, including altered pH andtemperature optimum, requirement for divalent cations for enzymaticactivity, mechanisms of enzymatic inhibition, substrate affinity andspecificity; localization upon secretion; localization signals;glycosylation sites; disulfide-bonds and unpaired cysteines;compatibility with in vitro expression systems; compatibility withfusion carriers; compatibility with purification methods; toxicologicalprofile; tissue penetration; pharmacokinetics; and pharmacodynamics. 55.The method of claim 54, wherein the DNASE variants are evaluated usingan in vitro nucleic acid degradation assay, which optionally employs oneor more of single or double-stranded DNA, plasmid DNA, mitochondrialDNA, NETs, or chromatin.
 56. The method of claim 55, wherein the assayis a NET-degrading assay.
 57. The method of claim 54, wherein the DNASEvariants are evaluated for their expression potential in prokaryoticand/or eukaryotic expression systems.
 58. The method of claim 54,wherein the DNASE variants are evaluated for short term and/or long termstability.
 59. The method of claim 54, wherein the DNASE variants areevaluated in animal models.
 60. The method of claim 59, wherein theDNASE variants are evaluated for immunogenic potential, half-life incirculation, protease resistance, bioavailability, and/or NET-degradingactivity.
 61. The method of claim 60, wherein the DNASE variants areevaluated in a disease models, which is optionally a rodent model or aprimate model.
 62. The method of claim 61, wherein the model is agenetically modified mouse deficient in D1 and D1L3 activity, the mousefurther having a heterologous expression of a G-CSF polynucleotide orinduction of a sustained endogenous G-CSF expression.
 63. The method ofany one of claims 1 to 62, wherein the selected DNASE variant isformulated for topical, parenteral, or pulmonary administration.
 64. Themethod of claim 63, wherein the selected DNASE variant is formulated forintradermal, intramuscular, intraperitoneal, intraarticular,intravenous, subcutaneous, intraarterial, oral, sublingual, pulmonary,or transdermal administration.
 65. A method for treating a subject inneed of extracellular DNA degradation, extracellular chromatindegradation, extracellular trap (ET) degradation and/or neutrophilextracellular trap (NET) degradation, the method comprises administeringa therapeutically effective amount of the DNASE variant formulated inaccordance with any one of claims 1 to
 64. 66. A DNASE variant orpharmaceutical composition thereof, the DNASE variant comprising one ormore building block substitutions and/or an N-terminal fusion of humanalbumin and a linker amino acid sequence.
 67. The DNASE variant of claim66, wherein the variant is a D1 variant comprising an amino acidsequence that is at least 80% identical to the enzyme defined by SEQ IDNO:
 1. 68. The DNASE variant of claim 67, wherein the D1 variant has oneor more mutations selected from non-human D1 enzymes, and which areselected from: K24R, I25M, Q31R, T32S, E35D, V44S, V44T, V44A, S45V,S45K, S45N, S45H, Q49K, Q49R, S52Q, S52R, R53L, I56V, A57V, L58V, V59I,S60T, T68V, D75N, N76E, N76K, N76T, N76E, N76Y, N76S, Q79R, Q79E, D80K,D80H, A81K, A81I, A81D, P82A, P82T, D83N, D83G, T84N, T84A, Y85F, H86R,Y87F, Y87H, V88I, V89I, V89A, N96K, N96R, N96S, S97T, R101Q, V105L,Y106F, D109S, Q110R, Q110K, A113V, A113I, S114L, S116T, Y108Q, Y108H,Y108L, P125S, N128T, T130S, N132S, N132A, A136S, I137V, R139K, F141S,F141H, S142C, R143P, R143H, F144Y, F144S, F144L, V147K, V147Q, R148Q,R148S, E149K, I152V, P154A, A157S, G160E, G160T, G160L, G160S, D161E,V163A, S164S, D167N, A168S, D175N, Q177W, Q177R, E178Q, E178K, E178H,G181D, G181H, E183Q, E183N, V185I, M186V, L187F, G194D, C195Y, R199T,R199A, R199S, P200S, P200A, P200T, P200L, Q202H, S204A, W209R, T210M,T210E, P212S, T213A, T213I, T213P, Q215K, Q215R, P219L, S221T, S221N,A226V, A226S, T227S, T227K, P228S, H230N, A232P, M241T, M241A, M241P,M241S, R244Q, G245D, G245A, G245H, G245R, G245S, D250N, D250S, D250E,D250G, L253V, L253A, L253M, N256D, A259V, A260E, Y261F, G262R, S264T,D265N, D265S, D265E, Q266E, L267M, L267T, Q269E, Q269L, M280T, M280A,K282R, K282A, K282T, K282insK, and K282insR.
 69. The DNASE variant ofclaim 67 or 68, wherein the D1 variant has a building block substitutionselected from human D1L1.
 70. The DNASE variant of claim 67 or 68,wherein the D1 variant has a building block substitution selected fromhuman D1L2.
 71. The DNASE variant of claim 67 or 68, wherein the D1variant has a building block substitution selected from human D1L3. 72.The DNASE variant of claim 67 or 68, wherein the D1 variant has abuilding block substitution selected from human D1L3-2.
 73. The DNASEvariant of claim 66, wherein the variant is a D1L1 variant comprising anamino acid sequence that is at least 80% identical to the enzyme definedby SEQ ID NO:
 2. 74. The DNASE variant of claim 73, wherein the D1L1variant further comprises one or more mutations selected from non-humanD1L1 enzymes, and which are selected from: A26T, Q27H, A32T, A32S, V34L,A35T, A35I, R36K, Q38S, Q38E, Q38H, Q38Y, Q38P, Q38D, M40K, M40L, T42I,L43F, R45Q, R45K, L47V, M53T, S61A, S62T, G63Q, G63D, G63N, G63S, 564N,S64A, S64K, S64T, A65T, P66L, P66S, L68F, R71Q, R71E, E72K, N74S, R75K,F76Y, D77K, D77Q, D77Y, D77G, G78A, G78S, G78N, G78D, G80R, G80K, P81S,P81F, P81C, S83R, T84F, T84S, L85H, S86N, S86K, P88S, P88D, Q89L, Q89M,S93N, 593G, T94A, M96V, M96K, T98K, V100A, F102I, H106D, K107R, K107E,K107R, T108A, Q109E, V110L, L111R, S112N, S112D, S112E, S113F, V115Q,V115L, V115M, N117D, N117E, N117P, N177S, E119T, E119Q, E119K, V122I,V122L, A124T, A130G, A130C, Q131H, Q131W, S133T, L134F, P135R, N137D,N137K, V138T, V138I, L142V, V143A, A153D, K156P, K156N, K156T, L159K,Y162H, D163E, D163T, E167D, V168A, S169Y, S169A, Q170R, Q170G, H171R,S174N, S174T, K175E, K175Q, S176N, V177M, V177I, A188T, T191A, T191N,D196K, D196N, D196S, D196A, D196G, E199L, E199A, E199V, E203K, E203D,E203Q, P204A, P204V, P204T, H207R, H207S, V209A, I210V, A211P, E214D,E241V, H223N, T225A, V229I, L231V, L231M, E234Q, E234V, R235G, R235T,R235L, C236L, R237Q, S238M, S238K, S238G, L240M, H241K, H241Q, H241S,H241R, T242A, T242S, T242N, T242G, A244T, D247N, T240K, T250R, Q250Q,S251T, S251R, Q252R, Q252G, T255N, T255S, E258Q, E259Q, N261R, N261K,M261T, I262V, E271D, K273S, K273N, K273D, K273A, L274del, S275Q, S275K,S275R, Q276A, A277T, A277V, H278P, H278Q, S279G, S279N, S279R, C279S,I280V, A280V, Q281P, Q281L, L283H, L283P, S284Y, S284C, S284H, S284G,T286A, T286S, T286V, V287T, V287A, F287F, G287V, L288A, L288S, L289S,L289V, L289M, S292L, S292P, S295P, S295T, S295A, P296S, Q297E, L298C,C299D, C299G, C299S, P300L, A301Q, A301V, and A302M.
 75. The DNASEvariant of claim 73 or 74, wherein the D1L1 variant has a building blocksubstitution selected from human D1.
 76. The DNASE variant of claim 73or 74, wherein the D1L1 variant has a building block substitutionselected from human D1L2.
 77. The DNASE variant of claim 73 or 74,wherein the D1L1 variant has a building block substitution selected fromhuman D1L3.
 78. The DNASE variant of claim 73 or 74, wherein the D1L1variant has a building block substitution selected from human D1L3-2.79. The DNASE variant of claim 73 or 74, wherein the D1L1 variantcontains one or more amino acid substitutions, additions, or deletionsin the C-terminal tail.
 80. The DNASE variant of claim 66, wheren thevariant is a D1L2 variant comprising an amino acid sequence that is atleast 80% identical to the enzyme defined by SEQ ID NO:
 3. 81. The DNASEvariant of claim 80, wherein the D1L2 variant further comprises one ormore mutations selected from non-human D1L2 enzymes, and which areselected from: L22K, I24V, I29V, S35N, S35H, S35R, S35T, V37A, S38L,A41D, A41V, A41G, G431, S44G, S44i, I45V, K48Q, L55I, L55V, A56T, A56M,P64A, 570D, 570T, A71T, A71L, A71S, A71V, M73L, E74Q, N77H, S78R, E81K,E81R, E83N, S85G, S85N, Q90E, Q90K, Q96H, F103Y, V104I, K107D, A109V,A109T, A109K, V110A, V113L, V113M, D114S, D114E, L117Q, P119S, E122G,V124A, V124F, S126N, E128D, F134V, A136V, A136T, G138S, G138R, T139S,T139C, S148C, A151P, P154A, A159P, A160G, A161P, A161T, Q162D, Q162K,Q162R, Q162T, N163K, N163E, L164V, L164F, I167V, H174N, Q175H, A178T,A178V, D192N, G195N, T196S, D198V, M199L, M199I, S210K, R213K, Q215H,A218P, A219S, E226Q, V227I, S243T, A252V, C253S, A255S, A255V, R256H,L257M, R259K, S260T, L261V, Q264H, T267S, T267A, D270N, G276D, G276S,T280S, T280D, T280A, A284C, I286V, L295F, F297S, F297T, F297P, H298R,and R299del.
 82. The DNASE variant of claim 80 or 81, wherein the D1L2variant has a building block substitution selected from human D1. 83.The DNASE variant of claim 80 or 81, wherein the D1L2 variant has abuilding block substitution selected from human D1L1.
 84. The DNASEvariant of claim 80 or 81, wherein the D1L2 variant has a building blocksubstitution selected from human D1L3.
 85. The DNASE variant of claim 80or 81, wherein the D1L2 variant has a building block substitutionselected from human D1L3-2.
 86. The DNASE variant of claim 80 or 81,wherein the D1L2 variant contains one or more amino acid substitutions,additions, or deletions in the proline-rich extension domain.
 87. TheDNASE variant of claim 66, wheren the variant is a D1L3 variantcomprising an amino acid sequence that is at least 80% identical to theenzyme defined by SEQ ID NO:
 4. 88. The DNASE variant of claim 87,wherein the D1L3 variant further comprises one or more mutationsselected from non-human D1L3 enzymes, and which are selected from: M21L,K22R, I22L, I22V, E33A, E33Y, E33G, S34A, S34T, Q36K, Q36R, E37A, E37Q,D48N, K39Q, K39H, K39R, K39C, N40E, N40Q, N40K, A41V, V44I, V53I, I53L,I54M, V57L, I60V, N64S, H64S, R66N, R66M, I70V, I70M, I70T, M72L, E73K,K74R, R77G, R81K, G82S, I83V, I83T, T84M, T84K, S91P, T91V, T91A, L105V,K107M, V111L, S112T, R115T, R115A, R115K, R115D, R115Q, S116K, S116N,S116Y, H118L, H118V, Y119F, H120G, Y122N, Q123E, D124A, D124S, D124N,G125E, A127V, A127T, V129A, F135Y, V137T, Q140H, S141A, H143F, H143Y,V146A, I152V, T157S, T160A, V162I, K163R, V169A, E170D, T173M, T173L,V175M, K176R, K176Q, H177S, H177R, R178Q, K180E, K180N, K181T, K181V,E183A, E183Q, A201S, K203Q, K203R, R212K, R212N, R212G, R212M, V214I,G218K, G218A, Q220E, Q220D, K227R, K227S, K227E, N239K, N239S, N239H,R239C, Q241P, E242D, E242N, V244I, S245N, S245R, K250R, K250D, K250R,K250G, K250N, K250Q, N252S, S253G, S253L, V254T, V254I, D256N, Q258R,Y261F, K262D, K262E, K262L, K262R, K262Q, T264S, E266S, E267K, E267Q,E267K, D270N, D270E, V271I, S282E, R285T, F287I, S290N, K291R, V294I,T295S, T295Q, L296V, L296P, L296S, R297K, K299R, T300K, T300A, S302G,S302A, S302V, S302T, K303N, K303S, K303R, R304H, R304S, S305P, S305T,and S305A.
 89. The DNASE variant of claim 87 or 88, wherein the D1L3variant has a building block substitution selected from human D1. 90.The DNASE variant of claim 87 or 88, wherein the D1L3 variant has abuilding block substitution selected from human D1L1.
 91. The DNASEvariant of claim 87 or 88, wherein the D1L3 variant has a building blocksubstitution selected from human D1L2.
 92. The DNASE variant of claim 87or 88, wherein the D1L3 variant contains one or more amino acidsubstitutions, additions, or deletions in the C-terminal tail.
 93. TheDNASE variant of claim 92, wherein the D1L3 variant contains one or moreamino acid substitutions, additions, or deletions in the internalsequence defined by SEQ ID NO: 11, and which is optionally deleted inwhole or in part.
 94. The DNASE variant of claim 66, wheren the variantis a D1L3-2 variant comprising an amino acid sequence that is at least80% identical to the enzyme defined by SEQ ID NO:
 5. 95. The DNASEvariant of claim 94, wherein the D1L3-2 variant further comprises one ormore mutations selected from non-human D1L3 enzymes, and which areselected from: M21L, K22R, I22L, I22V, E33A, E33Y, E33G, S34A, S34T,Q36K, Q36R, E37A, E37Q, D48N, K39Q, K39H, K39R, K39C, N40E, N40Q, N40K,A41V, V44I, V53I, I53L, I54M, V57L, I60V, N64S, H64S, R66N, R66M, I70V,I70M, I70T, M72L, E73K, K74R, R77G, V81L, S82T, R85T, R85A, R85K, R85D,R85Q, S86K, S86N, S86Y, H88L, H88V, Y89F, H90G, Y92N, Q93E, D94A, D94S,D94N, G95E, A97V, A97T, V99A, F105Y, V107T, Q110H, S111A, H113F, H113Y,V116A, I122V, T127S, T130A, V132I, K133R, V139A, E140D, T143M, T143L,V145M, K146R, K146Q, H147S, H147R, R148Q, K150E, K150N, K151T, K151V,E153A, E153Q, A171S, K173Q, K173R, R182K, R182N, R182G, R182M, V184I,G188K, G188A, Q190E, Q190D, K197R, K197S, K197E, N209K, N209S, N209H,R209C, Q211P, E212D, E212N, V214I, S215N, S215R, K220R, K220D, K220R,K220G, K220N, K220Q, N222S, S223G, S223L, V224T, V224I, D226N, Q228R,Y231F, K232D, K232E, K232L, K232R, K232Q, T234S, E236S, E237K, E237Q,E237K, D240N, D240E, V241I, S252E, R255T, F257I, S260N, K261R, V264I,T265S, T265Q, L266V, L266P, L266S, R267K, K269R, T270K, T270A, S272G,S272A, S272V, S272T, K273N, K273S, K273R, R274H, R274S, S275P, S275T,and S275A.
 96. The DNASE variant of claim 94 or 95, wherein the D1L3-2variant has a building block substitution selected from human D1. 97.The DNASE variant of claim 94 or 95, wherein the D1L3-2 variant has abuilding block substitution selected from human D1L1.
 98. The DNASEvariant of claim 94 or 95, wherein the D1L3-2 variant has a buildingblock substitution selected from human D1L2.
 99. The DNASE variant ofclaim 94 or 95, wherein the D1L3-2 variant contains one or more aminoacid substitutions, additions, or deletions in the C-terminal taildomain defined by SEQ ID NO:
 11. 100. The DNASE variant of claim 66,wheren the variant is a D2A variant comprising an amino acid sequencethat is at least 80% identical to the enzyme defined by SEQ ID NO: 6.101. The DNASE variant of claim 100, wherein the D2A variant furthercomprises one or more mutations selected from non-human D2A enzymes, andwhich are selected from: Q25R, L38H, L38N, R39S, R39T, G40S, G42R, E43D,A44T, A44K, A44V, A45P, A45T, R47K, R47N, R47S, Q50T, Q50M, Q50R, L54M,L54F, E56Q, S57N, S57H, S57E, G59D, G59E, G60D, R62Q, R62S, R65V, R65A,A66G, L67Y, L67H, L67F, L67S, N69D, P71S, P71K, P71T, E72D, E72T, V75L,R77L, Q80L, R84Q, S85K, S85N, T87S, T87N, L93V, Q101K, P102S, P102Y,S103R, K104S, K104E, K104G, A105S, Q106R, Q106K, D107H, S109T, M110G,M110S, M110N, R111H, H122Q, D123E, V129I, N134R, P137S, P138R, A139S,A142G, A143V, S145T, H148P, S149N, S149G, C151Q, C151R, T152K, Y153F,L158I, F162L, F164L, A165T, A165S, S168A, S168P, S168L, K169R, K169G,K169D, K169N, M170I, G171S, K172R, W180L, W180M, N183D, Y184H, Q185K,Q185R, I180F, I180D, Q192R, E193K, F194L, D196Y, N199T, N199E, V201I,V201T, G203N, G203Q, S207L, S207R, Q208H, Q208R, E209G, I215V, T216I,Q220R, Q220K, A221K, A223T, V224T, V224S, F231C, S232G, K233N, A244S,A245E, T249S, N250T, H257Q, H257P, T259S, V260P, V260S, V260A, D269G,I270A, I270T, I270V, W271Y, W271H, W271Q, Q272K, Q272H, V273I, L274F,N275D, N277T, Q278E, I279T, A280G, A285S, G286R, P287L, S288T, S288A,S288N, N290S, S291A, S301A, S301T, K303Q, K303E, G304R, T307A, T307V,Q316K, G317A, G317R, E319T, Q320H, L326V, A328T, L330V, L330M, A332S,L333F, Q338R, Q338K, P339S, N343D, N343A, Y344W, Y344C, Q345K, andQ345E.
 102. The DNASE variant of claim 100 or 101, wherein the D2Avariant has a building block substitution selected from human D2B. 103.The DNASE variant of claim 66, wheren the variant is a D2B variantcomprising an amino acid sequence that is at least 80% identical to theenzyme defined by SEQ ID NO:
 7. 104. The DNASE variant of claim 103,wherein the D2B variant further comprises one or more mutations selectedfrom non-human D2B enzymes, and which are selected from: A28P, A28T,T29E, T29V, T29K, S31A, R33I, N34S, E36Y, E36D, A37P, T44I, T44A, T44V,K50R, R51Q, R51K, Q52T, N53S, N53D, N53E, K54R, E55A, E55G, S56G, G57E,G57T, G57R, T59A, T59M, E62Q, E62D, E62G, T70R, T70M, T701, R71Q, S72T,R74N, R74S, R74K, K75R, E77L, E77H, E77K, Q78Y, Q78H, Q78L, M80I, M80V,D82T, D82S, D82A, T83S, K84R, K84D, V86A, V86S, Q92E, Q93H, E96D, A97T,Y98H, Y98N, Y98C, A99D, A99H, S100A, S100F, K101E, S102T, S102N, S102D,N104D, N104S, L108V, I109L, G113A, V114I, K116G, K116A, P117S, V118A,N119T, N119G, N119S, Y120C, R122G, K123Q, K123N, Y124F, T127A, L132V,V136T, V136I, I145V, Q147K, Q147R, I151V, I151T, E154H, E154K, D157E,P160T, P160S, T161S, R164Q, N165Y, N165H, G166A, S168T, S168A, S168N,I170L, I170M, F174L, K175G, K175R, N178S, Y179F, A181E, A181T, S184F,V188I, C189L, C189F, C189Y, N190Q, V192I, S195R, S197F, A200S, A200N,A200T, T201I, T201A, H203R, Q204W, Q204M, E205K, I207V, I207F, H208Y,H208Y, M209L, Q211R, L212M, T214A, R215K, R215G, A216S, S217T, S217H,S218A, S219L, E220K, G223V, G223S, R224Q, L225Y, L225R, L225H, T227A,T228E, T228V, T228S, Q230H, Q233R, Q235L, K236N, K236S, L238V, L238I,S243F, D244S, D244T, S245F, F246Y, L247T, L247H, A252T, A252V, A253G,M255I, R258K, R258H, R258Q, T261V, T265A, T265V, E266Q, T267S, R270K,R272K, R272N, R272G, Q273H, Y283H, C285I, I288V, A290S, K292G, K292R,L293V, L293G, L293I, R295G, R295S, R295L, R295H, H296K, H296Q, Y298D,S300P, Y302R, Y302H, Q303H, A306S, I310V, Q312I, Q312T, Q312R, Q312L,G314D, G314R, T315S, K316A, K316Q, N317A, R318H, P329L, H330Y, F333L,F333S, S335G, T341S, T341N, Q342K, W344H, W344R, W344Q, Q345H, Q345Y,Q345R, Q345N, Q349H, Q351H, Q351D, Q351E, G352K, G352R, V354Y, L355S,Y356R, Y356H, Y357H, E358G, E358A, S359F, S359N, S359D, and K361N. 105.The DNASE variant of claim 103 or 104, wherein the D2B variant has abuilding block substitution selected from human D2A.
 106. The DNASEvariant of any one of claims 66 to 105, wherein the variant comprises anN-terminal fusion to a human albumin amino acid sequence, and a linkeramino acid sequence, which is optionally composed of Gly and Ser. 107.The DNASE variant of any one of claims 66 to 105, wheren the variantcomprises at least one building block substitution and a half-lifeextending moiety independently selected from albumin, transferrin, Fc,and elastin-like protein.
 108. The DNASE variant of any one of claims 66to 105, wherein the variant comprises one or more polyethylene glycol(PEG) moieties.
 109. The DNASE variant of any one of claims 66 to 108,wherein the DNASE variant is formulated for topical, parenteral, orpulmonary administration.
 110. The DNASE variant of claim 109, whereinthe DNASE variant is formulated for intradermal, intramuscular,intraperitoneal, intraarticular, intravenous, subcutaneous,intraarterial, oral, sublingual, pulmonary, or transdermaladministration.
 111. A method for treating a subject in need ofextracellular DNA degradation, extracellular chromatin degradation,extracellular trap (ET) degradation and/or neutrophil extracellular trap(NET) degradation, the method comprises administering a therapeuticallyeffective amount of the DNASE variant or pharmaceutical compositionthereof of any one of claims 66 to 110.